Fully human antibodies to high molecular weight-melanoma associated antigen and uses thereof

ABSTRACT

Disclosed herein are isolated human monoclonal antibodies, and functional fragments thereof, that specifically bind HMW-MAA. Nucleic acids encoding these antibodies, expression vectors including these nucleic acid molecules, and isolated host cells that express the nucleic acid molecules are also disclosed. The antibodies can be used to detect HMW-MAA in a sample. Methods of diagnosing cancer, or confirming a diagnosis of cancer, are disclosed herein that utilize these antibodies. Methods of treating a subject with cancer are also disclosed.

PRIORITY CLAIM

This application is the U.S. National Stage of International ApplicationNo. PCT/US2009/060903, filed Oct. 15, 2009, which was published inEnglish under PCT Article 21(2), which claims the benefit of U.S.Provisional Application No. 61/106,055, filed Oct. 16, 2008, which isincorporated herein by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under contract numbersCA16056 and CA105500, awarded by the National Cancer Institute of theNational Institutes of Health. The government has certain rights in theinvention.

FIELD

This disclosure concerns fully human monoclonal antibodies, particularlyhuman monoclonal antibodies that specifically bind high molecularweight-melanoma associated antigen (HMW-MAA), and their use.

BACKGROUND

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells (“Cellular andMolecular Immunology” (1991) (eds.) Abbas A. K., Lechtman, A. H., Pober,J. S.; W.B. Saunders Company, Philadelphia: pages 340-341). Melanomasmake up approximately three percent of all skin cancers and theworldwide increase in melanoma is unsurpassed by any other neoplasm withthe exception of lung cancer in women (“Cellular and MolecularImmunology” (1991) (eds.) Abbas, A. K., Lechtiman, A. H., Pober, J. S.;W.B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala(1993) Principles and Practice of Oncology 7:1-16). Even when melanomais apparently localized to the skin, up to 30% of the patients willdevelop systemic metastasis and the majority will die (Kirkwood andAgarwala (1993) Principles and Practice of Oncology 7:1-16). Classicmodalities of treating melanoma include surgery, radiation andchemotherapy. In the past decade, immunotherapy and gene therapy haveemerged as new and promising methods for treating melanoma.

Strong evidence that an immune response to cancer exists in humans isprovided by the existence of lymphocytes within melanoma deposits. Theselymphocytes, when isolated, are capable of recognizing specific tumorantigens on autologous and allogeneic melanomas in a majorhistocompatibility complex (MHC)-restricted fashion (Itoh et al. (1986),Cancer Res. 46: 3011-3017; Muul et al. (1987), J. Immunol. 138:989-995);Topalian et al. (1989) J. Immunol. 142: 3714-3725; Darrow et al. (1989)J. Immunol. 142: 3329-3335; Hom et al. (1991) J. Immunother. 10:153-164;Kawakami et al. (1992) J. Immunol. 148: 638-643; Hom et al. (1993) J.Immunother. 13:18-30; O'Neil et al. (1993) J. Immunol. 151: 1410-1418).Tumor infiltrating lymphocytes (TIL) from patients with metastaticmelanoma recognize shared antigens including melanocyte-melanoma lineagespecific tissue antigens in vitro (Kawakami et al. (1993) J. Immunother.14: 88-93; Anichini et al. (1993) J. Exp. Med. 177: 989-998).Anti-melanoma T cells appear to be enriched in TIL probably as aconsequence of clonal expansion and accumulation at the tumor site invivo (Sensi et al. (1993) J. Exp. Med. 178:1231-1246). The fact thatmany melanoma patients mount cellular and humoral responses againstthese tumors and that melanomas express both MHC antigens and tumorassociated antigens (TAA) suggests that identification andcharacterization of additional melanoma antigens will be important forimmunotherapy of patients with melanoma.

The human chondroitin sulfate proteoglycan HMW-MAA, also know as CSPG4,is an early cell surface melanoma progression marker implicated instimulating tumor cell proliferation, migration and invasion. Clinicalstudies have indicated HMW-MAA as a relevant therapeutic target becausea vaccine targeting HMW-MAA in patients with melanoma immunized withHMW-MAA mimics provides significant survival advantage only to a subsetof patients who developed HMW-MAA-specific antibodies, but not to thosepatients who did not develop them. In addition, the biologicsignificance of targeting HMW-MAA could be related to the role inregulating cell growth and differentiation. However, a need remains forother immunotherapeutic strategies that target this antigen.

SUMMARY

Provided herein are fully human monoclonal antibodies that specificallybind HMW-MAA. Functional fragments of fully human monoclonal antibodiesare also provided. In some embodiments, the human monoclonal antibodiesare single chain variable fragments (scFv). Further provided arecompositions including the HMW-MAA-specific antibodies and functionalfragments thereof, nucleic acids encoding these antibodies, expressionvectors comprising the nucleic acids, and isolated host cells thatexpress the nucleic acids.

Also provided are immunoconjugates comprising the human monoclonalantibodies that specifically bind HMW-MAA. Compositions comprising theimmunoconjugates are also provided.

The antibodies and compositions provided herein can be used for avariety of purposes, such as for confirming the diagnosis of cancer in asubject. Thus, provided herein is a method of confirming the diagnosisof cancer in a subject, that includes contacting a sample from thesubject diagnosed with cancer with a human monoclonal antibody thatspecifically binds HMW-MAA, and detecting binding of the antibody to thesample. An increase in binding of the antibody to the sample relative tobinding of the antibody to a control sample confirms the cancerdiagnosis. In some embodiments, the method further comprises contactinga second antibody that specifically recognizes the HMW-MAA-specificantibody with the sample, and detecting binding of the second antibody.

Similarly, provided herein is a method of detecting cancer in a subjectthat includes contacting a sample from the subject with a humanmonoclonal antibody described herein, and detecting binding of theantibody to the sample. An increase in binding of the antibody to thesample relative to a control sample detects cancer in the subject. Insome embodiments, the methods further comprise contacting a secondantibody that specifically recognizes the HMW-MAA-specific antibody withthe sample, and detecting binding of the second antibody.

Further provided is a method of treating a subject diagnosed withcancer, that includes administering to the subject a therapeuticallyeffective amount of a HMW-MAA-specific monoclonal antibody, a functionalfragment thereof, or an immunoconjugate comprising the antibody orfunctional fragment thereof. In some examples, the cancer is a melanoma,a head and neck cancer or a glioma.

Also provided are HMW-MAA peptide mimics The peptide mimics disclosedherein bind a human monoclonal antibody specific for HMW-MAA. In someembodiments, the peptide mimics comprise the consensus sequencePXXYXPXXD (SEQ ID NO: 9).

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Selective reactivity in ELISA of soluble scFv C21 with HMW-MAA⁺human cell lines. Cultured human melanoma cells Colo38, FO-1, SK-MEL-28and Melur, all of which express HMW-MAA, and human prostate carcinomacells PC3, human bladder carcinoma cells T24, human breast carcinomacells T47D and human B lymphoid cells JY, LG-2 and LKT13, all of whichdo not express HMW-MAA and rat neuroblastoma cells B49, which express aHMW-MAA homolog were incubated at 4° C. for 2 h with 50 μl of SNT scFvC21 (

) and with biotinylated mAb 9E10 (2.5 μg/ml 1% BSA-PBS). Binding of scFvfragments was detected using SA-HRP. Results are expressed as absorbanceat 490 nm. Human anti-anti-id scFv #119 (▬) was used as a specificitycontrol.

FIG. 2. Flow cytometry analysis of HMW-MAA⁺ M14#5/HMW-MAA melanoma cellsstained with scFv C21. M14#5/HMW-MAA transfectants and parental HMW-MAA⁻M14#5 cells were incubated on ice with PP scFv C21 and mAb 9E10 (emptyhistogram) and control PP scFv 119 and mAb 9E10 (grey histogram) (toppanel), with HMW-MAA-specific mouse mAb 763.74 (0.5 μg) (emptyhistogram) and control mAb MK2-23 (grey histogram) (middle panel) andwith HLA class I antigen-specific mAb TP25.99 (empty histogram) andcontrol mAb MK2-23 (grey histogram) (bottom panel). Binding ofantibodies was detected using RPE-labeled F(ab′)₂ fragments of goatanti-mouse Ig antibodies. Cells were analyzed with a FACScan™ flowcytometer. Results are expressed as fluorescence intensity.

FIG. 3. Structural relationship between molecules recognized by scFv C21and by HMW-MAA-specific mouse mAb 763.74 in a HMW-MAA⁺ Colo38 celllysate. A 1% NP-40 extract of ¹²⁵I-labeled Colo38 cells wasimmunodepleted with mAb 763.74. The immunodepleted cell extract wasimmunoprecipitated with insolubilized mAb 763.74 and scFv C21. Antigenswere eluted and analyzed by SDS-PAGE in an 8% polyacrylamide gel. Gelswere fixed, dried and autoradiographed for up to 1 day at −80° C. A 1%NP-40 extract of ¹²⁵I-labeled Colo38 cells immunodepleted with 100 kDMAA-specific mouse mAb 376.94 was used as a control.

FIG. 4. Role of N-linked glycosylation in the expression of theantigenic determinant recognized by scFv C21 on HMW-MAA isolated from aHMW-MAA⁺ Colo38 cell extract. A 1% NP-40 extract of Colo38 cellslabelled with ³⁵S-methionine in the presence of tunicamycin (0, 2 and 3μg/ml,) was immunoprecipitated with scFv C21. Antigens were eluted fromthe immunoadsorbent and analyzed by SDS-PAGE in an 8% polyacrylamidegel. Gels were fixed, dried and processed for fluorography for up tothree days at −80° C. using Hyperfilm-ECL. HMW-MAA-specific mouse mAb763.74 was used as a control.

FIG. 5. Spatial proximity of the determinant defined by scFv C21 and ofthat defined by mouse mAb VF1-TP34 on HMW-MAA⁺ melanoma cells SK-MEL-28.Varying concentrations of mAb VF1-TP34 (-●-) (left panel) were mixedwith biotinylated scFv C21 (0.25 μg/well). The mixture was thentransferred to wells containing HMW-MAA⁺ cells and incubated for 1 h at4° C. Binding of antibodies was detected using SA-HRP. Results areexpressed as % inhibition. Likewise, varying amounts of PP scFv C21(-▴-) (right panel) were mixed with biotinylated mAb VF1-TP34 (40ng/well). The mixture was then transferred to wells containing HMW-MAA⁺cells and incubated for 1 h at 4° C. The unrelated mAb TP25.99 (-∘-) andthe unrelated PP scFv 119 (-Δ-) were used as specificity controls.

FIG. 6. Immunohistochemical staining by scFv C21 of frozen surgicallyremoved human nevi and melanoma lesions. scFv C21 stains a frozen nevus(panel A), a frozen primary melanoma lesion (panel B) and a frozenmetastatic melanoma lesion (panel C). The specificity of the stainingwas monitored by staining the nevus (panel D), the primary (panel E) andthe metastatic (panel F) melanoma lesions with the unrelated scFv #119.

FIG. 7. Reactivity in ELISA of scFv C21 with synthetic peptides derivedfrom those isolated by panning the phage display peptide library X15with scFv C21. Ninety-six well plates were coated for 2 h at 37° C. withsynthetic peptides P1C21 (

), P2C21 (125 μM) (

), and P3C21 (125 μM) (

), in 0.25% glutaradehyde-PBS. Following blocking with PBS-1% BSA, wellswere incubated for 2 h at room temperature with 50 μl of PP scFv C21 atand 50 μl of biotinylated mAb 9E10 (2.5 μg/ml 1% BSA-PBS). Binding ofscFv antibody to peptides was detected by addition of SA-HRP. Resultsare expressed as absorbance at 490 μM. The human scFv #28 whichrecognizes an unrelated HMW-MAA determinant and the unrelated peptideMART-1 (AAGIGILTV) (

) were used as specificity controls.

FIG. 8. Inhibition of the reactivity of scFv C21 with HMW-MAA⁺ cellsColo38 by synthetic peptides P1C21 and P3C21 and by modified peptideP3C21. Varying concentrations of synthetic peptides P1C21 (-●-),P3C21(-▴-), P3A5 (-⋄-), P3V7 (-

-) and P3S10 (-▪-) were incubated for 2 h at 4° C. with 50 μl of PP scFv(1:1600 dilution in 1% BSA-PBS) and 50 μl of biotinylated mAb 9E10 (2.5μg/ml 1% BSA-PBS). The mixture was then transferred to wells containingcells Colo38. Binding of scFvC21 was detected using SA-HRP (top panel).Results are expressed as % inhibition. The unrelated peptide MART-1(-∇-) and the scFv #28 (bottom panel) which recognizes an unrelatedHMW-MAA determinant were used as specificity controls.

FIG. 9. Development of antibodies with selective reactivity withHMW-MAK⁺ cells Colo38 in BALB/c mice immunized with peptide P1C21 andboosted with Colo 38 cells. Mice were immunized with KLH-conjugatedpeptide P1C21 (

) (50 μg/injection) on day 0, 21, 42, 63, 84 and 105, and with HMW-MAA⁺Colo38 melanoma cells (5×10⁵ cells/injection) on day 132. Sera wereharvested one week before the first immunization, and one week aftereach immunization. Two-fold dilutions of sera (100 μl/well) wereincubated with HMW-MAA⁺ Colo38 melanoma cells (1×10⁵/well). Following anadditional incubation with HRP-conjugated goat anti-mouse IgGantibodies, the reaction was developed using TMB substrate. O.D. wasmeasured at 450 nm Results are expressed as the mean±SD of the highestdilution of sera giving 50% of the maximal binding to HMW-MAA⁺ cells.Sera from mice immunized with peptide MB1₁₉₄₋₂₀₈ were used as controls.*p<0.05.

FIG. 10. Flow cytometry analysis of HMW-MAA⁺ M14#5/HMW-MAA melanomacells stained with antibodies elicited by peptide P1C21 and HMW-MAA⁺cells Colo38 in BALB/c mice. M14#5/HMW-MAA transfectants and parentalHMW-MAA⁻ M14#5 cells were incubated on ice with 100 μl of sera (1:60dilution) from mice immunized with peptide P1C21 (top panel), and withsera from mice immunized with peptide P1C21 or control peptideMB1₁₉₄₋₂₀₈ and boosted with HMW-MAA⁺ cells Colo38 (middle panel).Following washing, cells were incubated on ice with RPE-labeled F(ab′)₂fragments of goat anti-mouse Ig antibodies. Cells were then analyzedwith a FACScan™ flow cytometry. Results are expressed as fluorescenceintensity (empty histogram). Preimmune sera (grey histogram), sera frommice immunized with control peptide MB1₁₉₄₋₂₀₈ (top panel), sera frommice immunized with peptide MB1₁₉₄₋₂₀₈ and boosted with HMW-MAA⁺melanoma cells Colo38 (middle panel) and sera from mice immunized withpeptide P1C21 or peptide MB1₁₉₄₋₂₀₈ and boosted with HMW-MAA⁻ lymphoidcells LG2 (bottom panel) were used as controls.

FIG. 11. Immunochemical characterization of the HMW-MAA specificity ofsera from mice immunized with peptide P1C21 and boosted with HMW-MAA⁺cells Colo38. A 1% Triton X-100 extract of ¹²⁵I-labeled Colo38 melanomacells was immunoprecipitated with sera from mice sequentially immunizedwith peptide P1C21 and with HMW-MAA⁺ cells Colo38 (lane B). Antigenswere eluted from the immunoadsorbent, and analyzed by SDS-PAGE in an 8%polyacrylamide gel. Gels were fixed, dried and autoradiographed for 2days at −80° C. HMW-MAA-specific mouse mAb 763.74 (lane A), sera frommice immunized with peptide P1C21 and boosted with HMW-MAA⁻ lymphoidcells LG2 (lane C) and sera from mice immunized with control peptideMB1₁₉₄₋₂₀₈ and boosted with HMW-MAA⁺ Colo38 melanoma cells lane (lane D)were used as controls.

FIG. 12. DTH reaction to HMW-MAA⁺ melanoma cells Colo38 in BALB/c miceimmunized with peptide P1C21. Irradiated (20K rads) HMW-MAA⁺ cellsColo38 (5×10⁵ cells/mouse) (

) and HMW-MAA⁻ LG2 cells (5×10⁵ cells/mouse) (□) were injected on day132 into the right and left hind footpad, respectively, of BALB/c mice,which had been immunized six times with peptide P1C21 on days 0, 21, 42,63, 84 and 105. Amount of swelling induced by the injected cells wasmeasured and calculated by subtracting the thickness of the footpadmeasured at time 0 h from that measured 24 h after the injection ofcells. Mice immunized with control peptide MB1₁₉₄₋₂₀₈ using the sameschedule were injected at the same time point with either Colo38 or LG2cells, and used as controls. *p<0.05.

FIG. 13. Molecular model of scFv C21-P1C21 and -P3C21 peptide complex.V_(H) and V_(L) of scFv C21 are shown. The HCDR3 and LCDR3, which adoptnon-canonical structures, are also shown in. Binding of the cyclic P1C21peptide is shown in the left panel. The binding energy is −93.8Kcal/mol. The residues 7Pro, 5Tyr and 4Trp define the specificity. Thebinding of linear P3C21 to scFv C21 is shown in the right panel. Thebinding energy is −7.3 Kcal/mol. The P3C21 peptide adopts a moreextended structure in the binding mode. The critical contacts wereobserved between H1 and H3 through 4Arg, 7Pro and 5Tyr. One of theconserved residues (10Asp) is solvent exposed, but proximal leucinesburied in a hydrophobic pocket facilitate a stable complex.

FIG. 14. Flow analysis showing surface staining of human melanoma celllines with scFv-Fc C21.

FIG. 15. Flow analysis showing surface staining of human glioma celllines LN444 and A1207 with scFv-Fc C21 and surface staining of humanhead and neck cancer cell lines PCI 30 and PCI 13 with scFv-Fc C21.

FIGS. 16A and 16B. Digital images and graph showing the inhibition ofexperimental lung metastases of human melanoma cells MV3 by scFv-Fc C21.The protocol was as follows: Day 0: inject MV3 cells 1.4×10⁶/mouse i.v.;Day 3: Starting mAb therapy: 100 μg/mous, i.v. twice per week.; Day 27:Sacrifice mice and collect lung-formalin fixed and paraffin embedded andH&E stained for the following analysis:

Taking pictures of randomly selected 5 high power fields (×200) of eachsection and then measure the tumor area using the SPOT software. Thevalues shown are the mean tumor area of each group. ** indicates p value<0.01.

FIGS. 17A and 17B. Digital images and graph showing scFv-Fc C21 inhibitstumor cell proliferation in metastatic lesions in mice. The protocol wasas follows: Day 0: inject MV3 cells 1.4×10⁶/per mouse i.v.; Day 3:starting antibody therapy: 100 μg/per mouse, i.v. twice per week; Day27: sacrifice mice and collect lung-formalin fixed and paraffinembedded.

FIG. 18. Inhibition of primary tumor growth and local tumor recurrenceby scFv-Fc C21. The protocol was as follows: Day 0: S.C. inoculation oftumor cells MV3 1.5×10⁶/per mouse; Day7: i.v. scFv-Fc (100 μg)administration, 2× weekly; Day 28: surgical removal of tumor; Day 35:i.v. scFv-Fc (100 μg) administration, 2× weekly; Day 61: sacrifice ofmice due to the size of recurred tumor reached to the limit set byIACUC.

FIG. 19. Digital image showing inhibition of local tumor recurrence byscFv-Fc C21 in mice.

FIG. 20. Inhibition of post surgery spontaneous lung metastasis in miceby scFv-Fc C21.

FIG. 21 Digital images and graphs showing decreased PKC-α and p-Srclevels in surgically removed primary MV3 tumor tissues.

FIG. 22. Digital image and graph showing the inhibition of MDA-MB-231 invitro cell migration by scFv-Fc C21. MDA-MB-231 cells were seeded andincubated with either scFv-Fc C21, control scFv-Fc 119 or PBS in amigration assay. The pictures were taken under Zeiss InvertedFluorescence Microscope (AxioVision Software) of each well (×200). Theresults are expressed as % inhibition of migration, utilizing the valuesobtained in PBS without antibody as a reference. The values shown arethe mean of three independent experiments. *** indicates p<0.001.

FIG. 23. Graph of inhibition of MDA-MB-231 in vitro cell growth byscFv-Fc. C21. MDA-MB-231 cells were treated either with scFv-Fc C21 orcontrol scFv-Fc 119 in a 3-D (matrigel) setting for 6 days. The PBS,which was used as the solvent for both antibodies, was used as areference for 100% cell growth. Cells in each well were then harvestedfrom matrigel using Cell Recovery Solution (BD Pharmingen) and countedusing Trypan Blue by two individuals. The results are expressed as %inhibition of cell growth, utilizing the values obtained in PBS only asa reference. The values shown are the mean of two independentexperiments. *** indicates p<0.001

FIG. 24. Digital images of Western blots. The levels of p-Akt, p-Erk1/2and Erk1/2 are significantly decreased following treatment with scFv-FcC21.

FIG. 25. Digital images of a Western blot. β-Catenin is decreasedfollowing treatment with scFv0Fc C21. β-Catenin is involved in cell-celladhesion, cell signaling and gene transcription that are disruptedduring malignant transformation. The oncogenic effect of Notch1 onprimary melanoma cells was mediated by β-catenin, which was upregulatedfollowing Notch1 activation Inhibiting β-catenin expression reversedNotch1-enhanced tumor growth and metastasis (see also Klara B. et al.,J. Clin. Invest. 115(11): 3166-3176, 2005).

FIG. 26. Digital images of Western blots. The levels of p-PTEN(ser380/Thr382/383), non-phospho PTEN (ser380/Thr382/383), and PTEN areincreased following treatment with scFv-Fc C21. The levels ofphosphoinositide-dependent protein kinase (PDK1) are decreased followingtreatment with scFv-Fc C21. The levels of PKCα, p-FAK, and FAK aredecreased.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Apr. 6, 2011, 10.4 KB, which is incorporated byreference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of the V_(H) domain of scFv C21.

SEQ ID NO: 2 is the nucleotide sequence of the scFv C21 linker.

SEQ ID NO: 3 is the nucleotide sequence of the V_(L) domain of scFv C21.

SEQ ID NO: 4 is the nucleotide sequence of an immunoglobulin heavy chainincluding the hinge, CH2 and CH3 domains.

SEQ ID NO: 5 is the amino acid sequence of the V_(H) domain of scFv C21.

SEQ ID NO: 6 is the amino acid sequence of the V_(L) domain of scFv C21.

SEQ ID NO: 7 is the amino acid sequence of peptide P1C21.

SEQ ID NO: 8 is the amino acid sequence of peptide P2C21.

SEQ ID NO: 9 is the amino acid consensus sequence of peptides that bindscFv C21.

SEQ ID NO: 10 is the amino acid sequence of peptide P3C21.

SEQ ID NO: 11 is the amino acid sequence of peptide P3A5.

SEQ ID NO: 12 is the amino acid sequence of peptide P3V7.

SEQ ID NO: 13 is the amino acid sequence of peptide P3S10.

SEQ ID NO: 14 is the amino acid sequence of HMW-MAA

SEQ ID NO: 15 is an exemplary nucleic acid sequence encoding HMW-MAA.

DETAILED DESCRIPTION

Because of its high expression on melanoma cells with limited intra- andinter-lesional heterogeneity in a large percentage of patients withmelanoma, and its restricted distribution in normal tissues (Ferrone etal., Radiolabeled Monoclonal Antibodies for Imaging and Therapy 152:55,1988), the human chondroitin sulfate proteoglycan HMW-MAA represents anattractive target to implement immunotherapy of melanoma (Spitler etal., Cancer Res 47:1717-1723, 1987; Mittelman et al., J Clin Invest86:2136-2144, 1990; Quan et al., J Clin Oncol 15:2103-2110, 1997). Likemost of the identified human tumor antigens, HMW-MAA is a self antigen.As a result, it is poorly immunogenic in patients with melanoma (Hambyet al., Cancer Res 47:5284-5289, 1997). To overcome this limitation,which hinders the application of HMW-MAA as an immunogen in patientswith melanoma, mouse anti-idiotypic (anti-id) monoclonal antibodies(mAb), which mimic HMW-MAA determinants defined by mouse mAb, have beenused to implement active specific immunotherapy in clinical trials(Mittelman et al., J Clin Invest 86:2136-2144, 1990; Quan et al., J ClinOncol 15:2103-2110, 1997; Mittelman et al., Proc Natl Acad Sci USA89:466-470, 1992; Pride et al., Clin Cancer Res 4:2363-2370, 1998).HMW-MAA mimics have been found to induce HMW-MAA specific humoralimmunity in about 60% of the immunized patients (Mittelman et al., ProcNatl Acad Sci USA 89:466-470, 1992). The association of this immunitywith regression of metastases in a few patients (Mittelman et al.,Cancer Res 54:415-421, 1994) and with a statistically significantsurvival prolongation (Mittelman et al., Proc Natl Acad Sci USA89:466-470, 1992) has stimulated interest in optimizing the immunizationstrategy with HMW-MAA mimics.

Thus far, the characterization of the antigenic profile of HMW-MAA andthe development of mimics have been restricted to determinantsrecognized by mouse mAb. Through the use of a large panel of mouse mAb,six distinct and spatially distant antigenic determinants have beenidentified on HMW-MAA (Campoli et al., Crit. Rev Immunol 24:267-296,2004). In addition, mimics of the antigenic determinants defined bymouse mAb have been developed and characterized in their immunogenicity(Luo et al., J Immunol 174:7104-7110, 2005). In contrast, only a limitednumber of HMW-MAA-specific human scFv antibodies have been isolated fromphage display scFv antibody libraries and shown to recognize antigenicdeterminants distinct from those defined by mouse mAb (Desai et al.,Cancer Res 58:2417-2425, 1998; Noronha et al., J Immunol 161:2968-2976,1998). Furthermore, no mimics of the antigenic determinants identifiedby HMW-MAA-specific human antibodies have been isolated and analyzed fortheir immunogenic properties. The lack of this information, which mayreflect the low association constants of the available HMW-MAA-specifichuman scFv antibodies (Desai et al., Cancer Res 58:2417-2425, 1998;Noronha et al., J Immunol 161:2968-2976, 1998), has a negative impact onthe optimization of immunization strategies with HMW-MAA mimics.

Thus, disclosed herein is a HMW-MAA-specific scFv (C21) isolated from asemi-synthetic phage display scFv antibody library which is morereflective of the human immune repertoire and is a source of scFvantibodies with a higher affinity than those previously described.Antibodies including one or more CDRs from this human monoclonalantibody that specifically bind HMW-MAA are also disclosed. Moreover,the fine specificity of scFv C21 is defined and utilized to isolateHMW-MAA peptide mimics from a phage display peptide library. Thesepeptide bind a human monoclonal antibody specific for HMW-MAA. In someembodiments, the peptide mimics comprise the consensus sequencePXXYXPXXD (SEQ ID NO: 9).

I. Abbreviations

CDR Complementarity determining region

CT Commuted tomography

DMSO Dimethyl sulfoxide

DTH Delayed type hypersensitivity

EDC N-ethyl-N′-(dimethylaminopropyl) carbodiimide

ELISA Enzyme-linked immunosorbent assay

FBS Fetal bovine serum

HMW-MAA High molecular weight melanoma associated antigen

HNSCC Head and neck squamous cell carcinoma

HPLC High pressure liquid chromatography

IPTG Isopropyl-β-D-thiogalactopyranoside

KLH Keyhole limpet haemocyanin

mAb Monoclonal antibody

MBS Maleimidobenzoyl-N-hydroxysuccinimide

MRI Magnetic resonance imaging

NHS N-hydroxysuccinimide

OD Optical density

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate-buffered saline

PET Positron emission tomography

PP Periplasmic preparation

s.c. Subcutaneous

scFv Single chain fragment variable

SDS Sodium dodecyl sulfate

SNT Supernatant

SPR Surface plasmon resonance

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand specifically binds an epitope of an antigen, such as HMW-MAA, or afragment thereof. Antibodies are composed of a heavy and a light chain,each of which has a variable region, termed the variable heavy (V_(H))region and the variable light (V_(L)) region. Together, the V_(H) regionand the V_(L) region are responsible for binding the antigen recognizedby the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′₂ fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies), heteroconjugate antibodies (suchas, bispecific antibodies). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs has been defined (see, Kabat et al., Sequencesof Proteins of Immunological Interest, U.S. Department of Health andHuman Services, 1991, which is hereby incorporated by reference). TheKabat database is now maintained online. The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species, such as humans. The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs in three-dimensionalspace.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds HMW-MAA will have aspecific V_(H) region and the V_(L) region sequence, and thus specificCDR sequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds HMW-MAA.

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. A “humanized” immunoglobulin is animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one embodiment, all the CDRs are from thedonor immunoglobulin in a humanized immunoglobulin. Constant regionsneed not be present, but if they are, they must be substantiallyidentical to human immunoglobulin constant regions, i.e., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin. A humanizedantibody binds to the same antigen as the donor antibody that providesthe CDRs. The acceptor framework of a humanized immunoglobulin orantibody may have a limited number of substitutions by amino acids takenfrom the donor framework. Humanized or other monoclonal antibodies canhave additional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In another embodiment,binding affinity is measured by ELISA. An antibody that “specificallybinds” an antigen, such as HMW-MAA with a high affinity and does notsignificantly bind other unrelated antigens.

Breast cancer: A neoplastic condition of breast tissue that can bebenign or malignant. The most common type of breast cancer is ductalcarcinoma. Ductal carcinoma in situ is a non-invasive neoplasticcondition of the ducts. Lobular carcinoma is not an invasive disease butis an indicator that a carcinoma may develop. Infiltrating (malignant)carcinoma of the breast can be divided into stages (I, IIA, IIB, IIIA,IIIB, and IV).

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treating alymphoma, leukemia, or another tumor. In one embodiment, achemotherapeutic agent is a radioactive compound. One of skill in theart can readily identify a chemotherapeutic agent of use (see forexample, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) ed., © 2000Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): OncologyPocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995;Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds): The CancerChemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).Combination chemotherapy is the administration of more than one agent totreat cancer. One example is the administration of an antibody thatbinds HMW-MAA or a fragment thereof used in combination with aradioactive or chemical compound.

Chimeric antibody: An antibody that includes sequences derived from twodifferent antibodies, which typically are of different species. Mosttypically, chimeric antibodies include human and murine antibodydomains, generally human constant regions and murine variable regions,murine CDRs and/or murine SDRs.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof an antibody to HMW-MAA. For example, a human antibody thatspecifically binds HMW-MAA can include at most about 1, at most about 2,at most about 5, and most about 10, or at most about 15 conservativesubstitutions and specifically bind the original HMW-MAA polypeptide.The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibody specifically binds HMW-MAA. Non-conservative substitutions arethose that reduce an activity or binding to HMW-MAA.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Complementarity determining region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to thecells intended to be targeted, as opposed to the cells of the rest of anorganism. In one embodiment, in contrast, the term “toxicity” refers totoxicity of an immunotoxin to cells other than those that are the cellsintended to be targeted by the targeting moiety of the immunotoxin, andthe term “animal toxicity” refers to toxicity of the immunotoxin to ananimal by toxicity of the immunotoxin to cells other than those intendedto be targeted by the immunotoxin.

Degenerate variant: A polynucleotide encoding a HMW-MAA polypeptide oran antibody that binds HMW-MAA that includes a sequence that isdegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore,all degenerate nucleotide sequences are included as long as the aminoacid sequence of the HMW-MAA polypeptide or antibody that binds HMW-MAAencoded by the nucleotide sequence is unchanged.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, melanoma, breast cancer, glioma,head and neck cancer or prostate cancer. Diagnostic methods differ intheir sensitivity and specificity. The “sensitivity” of a diagnosticassay is the percentage of diseased individuals who test positive(percent of true positives). The “specificity” of a diagnostic assay isone minus the false positive rate, where the false positive rate isdefined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” is theprobability of development (e.g., severity) of a pathologic condition,such as breast cancer or metastasis.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an anti-HMW-MAA antibody, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art (see, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985). Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc,¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I, fluorophores,chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such as HMW-MAA.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Framework region: Amino acid sequences interposed between CDRs.Framework regions include variable light and variable heavy frameworkregions. The framework regions serve to hold the CDRs in an appropriateorientation for antigen binding.

Glioma: A tumor composed of neuroglia in any developmental state.Gliomas include all intrinsic neoplasms of the brain and spinal cord,such as astrocytomas, ependymomas, and oligodendrogliomas. “Low-grade”gliomas are well-differentiated (not anaplastic); these are benign andportend a better prognosis for the patient. “High-grade” gliomas areundifferentiated or anaplastic; these are malignant and carry a worseprognosis.

HAMA (human anti-murine antibody) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the patient. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the patient.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody or functional fragment thereof. The effector molecule can be adetectable label or an immunotoxin. Specific, non-limiting examples oftoxins include, but are not limited to, abrin, ricin, Pseudomonasexotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin(DT), botulinum toxin, or modified toxins thereof, or other toxic agentsthat directly or indirectly inhibit cell growth or kill cells. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as the domain Ia of PE and the B chain ofDT) and replacing it with a different targeting moiety, such as anantibody. A “chimeric molecule” is a targeting moiety, such as a ligandor an antibody, conjugated (coupled) to an effector molecule. The term“conjugated” or “linked” refers to making two polypeptides into onecontiguous polypeptide molecule. In one embodiment, an antibody isjoined to an effector molecule. In another embodiment, an antibodyjoined to an effector molecule is further joined to a lipid or othermolecule to a protein or peptide to increase its half-life in the body.The linkage can be either by chemical or recombinant means. In oneembodiment, the linkage is chemical, wherein a reaction between theantibody moiety and the effector molecule has produced a covalent bondformed between the two molecules to form one molecule. A peptide linker(short peptide sequence) can optionally be included between the antibodyand the effector molecule. Because immunoconjugates were originallyprepared from two molecules with separate functionalities, such as anantibody and an effector molecule, they are also sometimes referred toas “chimeric molecules.” The term “chimeric molecule,” as used herein,therefore refers to a targeting moiety, such as a ligand or an antibody,conjugated (coupled) to an effector molecule.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence, such as an N-terminal repeat, such that the peptidewill bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”)response, or a B cell response (e.g. antibody production) against theantigen from which the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. In one specific non-limitingexample, an immunogenic polypeptide includes a region of HMW-MAA, or afragment thereof, wherein the polypeptide that is expressed on the cellsurface of a host cell that expresses the full-length HMW-MAApolypeptide.

Immunogenic composition: A composition comprising a polypeptide, such asa HMW-MAA polypeptide, that induces a measurable CTL response againstcells expressing HMW-MAA polypeptide, or induces a measurable B cellresponse (such as production of antibodies) against a HMW-MAApolypeptide. An immunogenic composition can also induce cytokineproduction. It further refers to isolated nucleic acids encoding aHMW-MAA polypeptide that can be used to express the HMW-MAA polypeptide(and thus be used to elicit an immune response against thispolypeptide). For in vitro use, an immunogenic composition may consistof the isolated protein or peptide epitope. For in vivo use, theimmunogenic composition will typically comprise the protein orimmunogenic peptide in pharmaceutically acceptable carriers, and/orother agents. Any particular peptide, such as a HMW-MAA polypeptide, ornucleic acid encoding the polypeptide, can be readily tested for itsability to induce a CTL or B cell response by art-recognized assays.Immunogenic compositions can include adjuvants, which are well known toone of skill in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopesImmunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, and pH) that are typicalinside a living mammal or a mammalian cell. While it is recognized thatsome organs are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionucleotides (such as ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F,^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I), fluorescentlabels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Linker: In some cases, a linker is a peptide within an antibody bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. “Linker” can alsorefer to a peptide serving to link a targeting moiety, such as anantibody, to an effector molecule, such as a cytotoxin or a detectablelabel.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule.The linkage can be either by chemical or recombinant means. “Chemicalmeans” refers to a reaction between the antibody moiety and the effectormolecule such that there is a covalent bond formed between the twomolecules to form one molecule.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Major histocompatibility complex (MHC): Generic designation meant toencompass the histocompatibility antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”). The term“motif” refers to the pattern of residues in a peptide of definedlength, usually about 8 to about 11 amino acids, which is recognized bya particular MHC allele. The peptide motifs are typically different foreach MHC allele and differ in the pattern of the highly conservedresidues and negative binding residues.

Melanoma: A form of cancer that originates in melanocytes (cells thatmake the pigment melanin). Melanocytes are found primary in the skin,but are also present in the bowel and eye. Melanoma in the skin includessuperficial spreading melanoma, nodular melanoma, acral lentiginousmelanoma, and lentigo maligna (melanoma). Any of the above types mayproduce melanin or can be amelanotic. Similarly, any subtype may showdesmoplasia (dense fibrous reaction with neurotropism) which is a markerof aggressive behavior and a tendency to local recurrence. Othermelanomas include clear cell sarcoma, mucosal melanoma and uvealmelanoma.

Features that affect prognosis are tumor thickness in millimeters(Breslow's depth), depth related to skin structures (Clark level), typeof melanoma, presence of ulceration, presence of lymphatic/perineuralinvasion, presence of tumor infiltrating lymphocytes (if present,prognosis is better), location of lesion, presence of satellite lesions,and presence of regional or distant metastasis. When melanomas havespread to the lymph nodes, one of the most important factors is thenumber of nodes with malignancy. The extent of malignancy within a nodeis also important; micrometastases in which malignancy is onlymicroscopic have a more favorable prognosis than macrometastases. Whenthere is distant metastasis, the five year survival rate is less than 10percent; the median survival is 6 to 12 months. Metastases to skin andlungs have a better prognosis. Metastases to brain, bone and liver areassociated with a worse prognosis.

Melanoma can be staged as follows:

-   -   Stage 0: Melanoma in Situ (Clark Level I), 100% Survival    -   Stage I/II: Invasive Melanoma, 85-95% Survival        -   T1a: Less than 1.00 mm primary, w/o Ulceration, Clark Level            II-III        -   T1b: Less than 1.00 mm primary, w/Ulceration or Clark Level            IV-V        -   T2a: 1.00-2.00 mm primary, w/o Ulceration    -   Stage II: High Risk Melanoma, 40-85% Survival        -   T2b: 1.00-2.00 mm primary, w/Ulceration        -   T3a: 2.00-4.00 mm primary, w/o Ulceration        -   T3b: 2.00-4.00 mm primary, w/Ulceration        -   T4a: 4.00 mm or greater primary w/o Ulceration        -   T4b: 4.00 mm or greater primary w/Ulceration    -   Stage III: Regional Metastasis, 25-60% Survival        -   N1: Single Positive Lymph Node        -   N2: 2-3 Positive Lymph Nodes OR Regional Skin/In-Transit            Metastasis        -   N3: 4 Positive Lymph Nodes OR Lymph Node and Regional            Skin/In Transit Metastases    -   Stage IV: Distant Metastasis, 9-15% Survival        -   M1a: Distant Skin Metastasis, Normal lactate dehydrogenase            (LDH)        -   M1b: Lung Metastasis, Normal LDH        -   M1c: Other Distant Metastasis OR Any Distant Metastasis with            Elevated LDH

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized and fullyhuman monoclonal antibodies. As used herein a monoclonal antibodyincludes antibody fragments, such as, but not limited to scFv, Fv, dsRv,or Fab.

Neoplasia, malignancy, cancer or tumor: The result of abnormal anduncontrolled growth of cells. Neoplasia, malignancy, cancer and tumorare often used interchangeably. The amount of a tumor in an individualis the “tumor burden” which can be measured as the number, volume, orweight of the tumor. A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue and/or canmetastasize is referred to as “malignant.” Examples of hematologicaltumors include leukemias, including acute leukemias (such as11q23-positive acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myelogenous leukemia and myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronicleukemias (such as chronic myelocytic (granulocytic) leukemia, chronicmyelogenous leukemia, and chronic lymphocytic leukemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer (including basal breast carcinoma,ductal carcinoma and lobular breast carcinoma), lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,medullary thyroid carcinoma, papillary thyroid carcinoma,pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladdercarcinoma, and CNS tumors (such as a glioma, astrocytoma,medulloblastoma, craniopharyogioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma and retinoblastoma).

In several examples, a tumor is melanoma, breast cancer, prostatecancer, glioma or a squamous cell carcinoma, such as head and neckcancer.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, bythe homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see for example, Current Protocols in Molecular Biology(Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (world wideweb ncbi.nlm.nib.gov). The BLASTN program (for nucleotide sequences)uses as defaults a word length (W) of 11, alignments (B) of 50,expectation (E) of 10, M=5, N=−4, and a comparison of both strands. TheBLASTP program (for amino acid sequences) uses as defaults a word length(W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix(see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (such as glycosylation orphosphorylation). In one embodiment, the polypeptide is HMW-MAApolypeptide. A “residue” refers to an amino acid or amino acid mimeticincorporated in a polypeptide by an amide bond or amide bond mimetic. Apolypeptide has an amino terminal (N-terminal) end and a carboxyterminal (C-terminal) end.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop,such as a reduction in tumor burden or a deacrease in the number of sizeof metastases. “Ameliorating” refers to the reduction in the number orseverity of signs or symptoms of a disease, such as cancer.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, 15 nucleotides or more inlength. Primers may be annealed to a complementary target DNA strand bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, and then extended along the target DNA strand by aDNA polymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, forexample, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. Both constitutive and inducible promotersare included (see for example, Bitter et al., Methods in Enzymology153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (such as the metallothioneinpromoter) or from mammalian viruses (such as the retrovirus longterminal repeat; the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) may be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used. A polynucleotide can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific nucleic acid sequences thatallow phenotypic selection of the transformed cells.

Prostate cancer: A type of cancer that forms in tissues of the prostate,a gland in the male reproductive system. Prostate cancer is classifiedas an adenocarcinoma, or glandular cancer, that begins when normalsemen-secreting prostate gland cells mutate into cancer cells. Theregion of prostate gland where the adenocarcinoma is most common is theperipheral zone. Initially, small clumps of cancer cells remain confinedto otherwise normal prostate glands, a condition known as carcinoma insitu or prostatic intraepithelial neoplasia (PIN). Although there is noproof that PIN is a cancer precursor, it is closely associated withcancer. Over time these cancer cells begin to multiply and spread to thesurrounding prostate tissue (the stroma) forming a tumor. Eventually,the tumor may grow large enough to invade nearby organs such as theseminal vesicles or the rectum, or the tumor cells may develop theability to travel in the bloodstream and lymphatic system. Prostatecancer is considered a malignant tumor because it is a mass of cellswhich can invade other parts of the body. Prostate cancer most commonlymetastasizes to the bones, lymph nodes, rectum and bladder.

Prostate cancer can be staged as follows:

-   -   Stage 0: No evidence of tumor    -   Stage I: Tumor present, but not detectable clinically or with        imaging        -   T1a: Tumor was incidentally found in less than 5% of            prostate tissue resected        -   T1b: Tumor was incidentally found in greater than 5% of            prostate tissue resected        -   T1c: Tumor was found in a needle biopsy performed due to an            elevated serum prostate specific antigen (PSA)    -   Stage II: The tumor can be felt (palpated) on examination, but        has not spread outside the prostate        -   T2a: The tumor is in half or less than half of one of the            prostate glands two lobes        -   T2b: The tumor is in more than half of one lobe, but not            both        -   T2c: The tumor is in both lobes    -   Stage III: The tumor has spread through the prostatic capsule        -   T3a: The tumor has spread through the capsule on one or both            sides        -   T3b: the tumor has invaded one or both seminal vesicles        -   Stage IV: The tumor has invaded other nearby structures

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation.

The HMW-MAA polypeptides disclosed herein, or antibodies thatspecifically bind HMW-MAA, can be purified by any of the means known inthe art. See for example Guide to Protein Purification, ed. Deutscher,Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, ProteinPurification: Principles and Practice, Springer Verlag, New York, 1982.Substantial purification denotes purification from other proteins orcellular components. A substantially purified protein is at least 60%,70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.

Recombinant toxins: Chimeric proteins in which a cell targeting moietyis fused to a toxin (Pastan et al., Science, 254:1173-1177, 1991). Ifthe cell targeting moiety is the Fv portion of an antibody, the moleculeis termed a recombinant immunotoxin (Chaudhary et al., Nature,339:394-397, 1989). The toxin moiety is genetically altered so that itcannot bind to the toxin receptor present on most normal cells.Recombinant immunotoxins selectively kill cells which are recognized bythe antigen binding domain. These recombinant toxins and immunotoxinscan be used to treat cancer, for example, a cancer in which HMW-MAA isexpressed.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a HMW-MAA polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity counted over the full lengthalignment with the amino acid sequence of the antibody using the NCBIBlast 2.0, gapped blastp set to default parameters. For comparisons ofamino acid sequences of greater than about 30 amino acids, the Blast 2sequences function is employed using the default BLOSUM62 matrix set todefault parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). Proteins with even greater similarity tothe reference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a HMW-MAA specific binding agent is an agent thatbinds substantially to a HMW-MAA polypeptide. An HMW-MAA specificbinding agent does not bind substantially to other unrelated proteins.In one embodiment, the specific binding agent is a human monoclonalantibody that specifically binds the HMW-MAA polypeptide.

The term “specifically binds” refers, with respect to an antigen such asHMW-MAA, to the preferential association of an antibody or other ligand,in whole or part, with a cell or tissue bearing that antigen and not tocells or tissues lacking that antigen. It is recognized that a certaindegree of non-specific interaction may occur between a molecule and anon-target cell or tissue. Nevertheless, specific binding may bedistinguished as mediated through specific recognition of the antigen.Although selectively reactive antibodies bind antigen, they may do sowith low affinity. On the other hand, specific binding results in a muchstronger association between the antibody (or other ligand) and cellsbearing the antigen than between the bound antibody (or other ligand)and cells lacking the antigen. Specific binding typically results ingreater than 2-fold, such as greater than 5-fold, greater than 10-fold,or greater than 100-fold increase in amount of bound antibody or otherligand (per unit time) to a cell or tissue bearing the HMW-MAApolypeptide as compared to a cell or tissue lacking the polypeptide.Specific binding to a protein under such conditions requires an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats are appropriate for selecting antibodies or otherligands specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

Squamous cell carcinoma: A type of cancer that originates in squamouscells, thin, flat cells that form the surface of the skin, eyes, variousinternal organs, and the lining of hollow organs and ducts of someglands. Squamous cell carcinoma is also referred to as epidermoidcarcinoma. One type of squamous cell carcinoma is head and neck headsquamous cell carcinoma (HNSCC). Head and neck squamous cell carcinomaincludes cancers of the nasal cavity, sinuses, lips, mouth, salivaryglands, throat and larynx.

HNSCC can be staged as follows:

Stage 0: No evidence of tumor.

Stage I: Tumor is 2 cm or less in greatest dimension; no evidence ofregional lymph node involvement or distant metastasis.

Stage II: Tumor is more than 2 cm, but no larger than 4 cm; no evidenceof regional lymph node involvement or distant metastasis.

Stage III: Tumor is larger than 4 cm; in some cases, the tumor hasspread to the lymph nodes; no evidence of distant metastasis.

Stage IV: Tumor has spread to the lymph nodes; in some cases, distantmetastases are present.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin,ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinumtoxin, saporin, restrictocin or gelonin, or modified toxins thereof. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as domain Ia of PE or the B chain of DT)and replacing it with a different targeting moiety, such as an antibody.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, Genbank Accession numbers and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present specification, including explanations of terms, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

III. Human Monoclonal Antibodies that Specifically Bind HMW-MAA

Disclosed herein are human monoclonal antibodies and functionalfragments thereof that specifically bind HMW-MAA. In one example,HMW-MAA has an amino acid sequence set forth as:

(SEQ ID NO: 14)EQMREEPEAA YRLIQGPQYG HLLVGGRPTS AFSQFQIDQG EVVFAFTNFS SSHDHFRVLALARGVNASAV VNVTVRALLH VWAGGPWPQG ATLRLDPTVL DAGELANRTG SVPRFRLLEGPRHGRVVRVP RARTEPGGSQ LVEQFTQQDL EDGRLGLEVG RPEGRAPGPA GDSLTLELWAQGVPPAVASL DFATEPYNAA RPYSVALLSV PEAARTEAGK PESSTPTGEP GPMASSPEPAVAKGGFLSFL EANMFSVIIP MCLVLLLLAL ILPLLFYLRK RNKTGKHDVQ VLTAKPRNGLAGDTETFRKV EPGQAIPLTA VPGQLFPSee also GENBANK® Accession No. AAI28111 incorporated herein byreference)

HMW-MAA is a human melanoma-associated chondroitin sulfate proteoglycanthat plays a role in stabilizing cell-substratum interactions duringearly events of melanoma cell spreading on endothelial basementmembranes. CSPG4 represents an integral membrane chondroitin sulfateproteoglycan expressed by human malignant melanoma cells.

HMW-MAA is also known as CSPG4. In vivo, it is present in a moleculethat consists of two noncovalently associated glycopolypeptides. One hasan apparent molecular weight of 280K, and the other has an apparentmolecular weight greater than 440K. HMW-MAA is synthesized and expressedby human melanoma cells (Spiro, R. C. et al. F. Biol. Chem. 264:1779(1989); Esko, J. D., et al., Science 241:1092, 1988). Proteoglycans areglycoproteins with glycosaminoglycan (GAG) polysaccharide chainscovalently attached to the serine residue in their core. The M+HMW-MAAcore protein is initially translated as a precursor with a molecularmass of 240K with asparagine N-linked oligosaccharides of the highmannose type.

In another example, the HMW-MAA is encoded by the nucleic acid sequenceset forth as:

(SEQ ID NO: 15)gggagcagat gagggaggag ccagaggcag cataccgcct catccaggga ccccagtatgggcatctcct ggtgggcggg cggcccacct cggccttcag ccaattccag atagaccagggcgaggtggt ctttgccttc accaacttct cctcctctca tgaccacttc agagtcctggcactggctag gggtgtcaat gcatcagccg tagtgaacgt cactgtgagg gctctgctgcatgtgtgggc aggtgggcca tggccccagg gtgccaccct gcgcctggac cccaccgtcctagatgctgg cgagctggcc aaccgcacag gcagtgtgcc gcgcttccgc ctcctggagggaccccggca tggccgcgtg gtccgcgtgc cccgagccag gacggagccc gggggcagccagctggtgga gcagttcact cagcaggacc ttgaggacgg gaggctgggg ctggaggtgggcaggccaga ggggagggcc cccggccccg caggtgacag tctcactctg gagctgtgggcacagggcgt cccgcctgct gtggcctccc tggactttgc cactgagcct tacaatgctgcccggcccta cagcgtggcc ctgctcagtg tccccgaggc cgcccggacg gaagcagggaagccagagag cagcaccccc acaggcgagc caggccccat ggcatccagc cctgagcccgctgtggccaa gggaggcttc ctgagcttcc ttgaggccaa catgttcagc gtcatcatccccatgtgcct ggtacttctg ctcctggcgc tcatcctgcc cctgctcttc tacctccgaaaacgcaacaa gacgggcaag catgacgtcc aggtcctgac tgccaagccc cgcaacggcctggctggtga caccgagacc tttcgcaagg tggagccagg ccaggccatc ccgctcacagctgtgcctgg ccagttattt cca

See also GENBANK® Accession No. BC128110, incorporated herein byreference. Once of skill in the art can readily use a nucleic acidsequence to produce a polypeptide, such as HMW-MAA using standard methodin molecular biology (see, for example, Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

Described herein are isolated human monoclonal antibodies and fragmentsthereof that specifically bind human HMW-MAA. In some embodiments, thehuman monoclonal antibody functional fragment is a scFv. Also describedare compositions including the provided human monoclonal antibodies orfunctional fragment thereof and a pharmaceutically acceptable carrier.Nucleic acids encoding these antibodies, expression vectors comprisingthese nucleic acids, and isolated host cells that express the nucleicacids are also provided.

Also described herein are immunoconjugates comprising the humanmonoclonal antibodies or functional fragment thereof that specificallybinds human HMW-MAA. The immunoconjugates can comprise any therapeuticagent, toxin or other moiety. In one example, the toxin is PE or avariant or fragment thereof. Compositions comprising theimmunoconjugates are also described.

Compositions comprising the human monoclonal antibodies thatspecifically bind HMW-MAA or functional fragment thereof can be used forscreening, research, detection and therapeutic purposes. For example,the human monoclonal antibodies or functional fragment thereof can beused to identify other antibodies that specifically bind HMW-MAA, suchas in competitive immunoassays.

Compositions comprising the human monoclonal antibodies thatspecifically bind HMW-MAA or functional fragment thereof can be used totreat a subject diagnosed with cancer, such as a cancer that exhibitsincreased expression of HMW-MAA relative to normal cells. For example,the antibodies can be used to treat melanoma, breast cancer, prostatecancer, ovarian cancer, colon cancer, stomach cancer, pancreatic cancer,glioma, chordoma, chondrosarcoma, glioma or a squamous cell carcinoma.Melanoma includes spreading melanoma, nodular melanoma, acrallentiginous melanoma, and lentigo maligna (melanoma). Squamous cellscarcinomas include, but are not limited to head and neck squamous cellcarcinoma, and squamous cell cancers of the skin, lung, prostate,esophagus, vagina and cervix.

Compositions comprising the HMW-MAA antibodies can also be used toprevent metastasis or decrease the number of micrometastases, such asmicrometastases to regional lymph nodes Immunoconjugates comprising theHMW-MAA antibodies also can be used to treat a patient diagnosed withcancer. The human monoclonal antibodies can also be used to diagnosecancer in a subject. For example, the human monoclonal antibodies can becontacted with a sample from the patient, such as a serum sample, todetect elevated levels of HMW-MAA. The antibodies and compositionsprovided herein can also be used to detect cancer in a subject or toconfirm the diagnosis of cancer in a patient.

Disclosed herein are fully human monoclonal antibodies that specificallybind human HMW-MAA and functional fragments thereof. A major limitationin the clinical use of mouse monoclonal antibodies is the development ofa human anti-murine antibody (HAMA) response in the patients receivingthe treatments. The HAMA response can involve allergic reactions and anincreased rate of clearance of the administered antibody from the serum.Various types of modified monoclonal antibodies have been developed tominimize the HAMA response while trying to maintain the antigen bindingaffinity of the parent monoclonal antibody. One type of modifiedmonoclonal antibody is a human-mouse chimera in which a murineantigen-binding variable region is coupled to a human constant domain(Morrison and Schlom, Important Advances in Oncology, Rosenberg, S. A.(Ed.), 1989). A second type of modified monoclonal antibody is thecomplementarity determining region (CDR)-grafted, or humanized,monoclonal antibody (Winter and Harris, Immunol. Today 14:243-246,1993). However, the antibodies disclosed herein are fully human; boththe framework region and the CDRs are derived from human sequences.Thus, a HAMA is not induced when these antibodies are administered to ahuman subject.

In some embodiments, the human monoclonal antibody or functionalfragment thereof comprises at least a portion of the heavy chain aminoacid sequence set forth as SEQ ID NO: 5 and specifically binds HMW-MAA.In some embodiments, the human monoclonal antibody or functionalfragment thereof comprises at least a portion of the light chain aminoacid sequence set forth as SEQ ID NO: 6 and specifically binds HMW-MAA.In some examples, the heavy chain of the antibody comprises amino acids27-38 of SEQ ID NO: 5 (CDR1), amino acids 56-65 of SEQ ID NO: 5 (CDR2),amino acids 105-115 of SEQ ID NO: 5 (CDR3), or a combination thereof. Insome examples, the heavy chain of the antibody comprises the heavy chainof the antibody comprises amino acids 27-38 of SEQ ID NO: 5 (CDR1),amino acids 56-65 of SEQ ID NO: 5 (CDR2), and amino acids 105-115 of SEQID NO: 5 (CDR3). In some examples, the light chain of the antibodycomprises amino acids 27-38 of SEQ ID NO: 6 (CDR1), amino acids 56-65 ofSEQ ID NO: 6 (CDR2), amino acids 105-110 of SEQ ID NO: 6 (CDR3), or acombination thereof. In some examples, the light chain of the antibodycomprises amino acids 27-38 of SEQ ID NO: 6 (CDR1), amino acids 56-65 ofSEQ ID NO: 6 (CDR2), and amino acids 105-110 of SEQ ID NO: 6 (CDR3). Insome embodiments, the human monoclonal antibody is labeled. In someexamples, the label is a fluorescence, enzymatic, or radioactive label.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁ or an IgG₂.The class of an antibody that specifically binds HMW-MAA can be switchedwith another. In one aspect, a nucleic acid molecule encoding V_(L) orV_(H) is isolated using methods well-known in the art, such that it doesnot include any nucleic acid sequences encoding the constant region ofthe light or heavy chain, respectively. The nucleic acid moleculeencoding V_(L) or V_(H) is then operatively linked to a nucleic acidsequence encoding a C_(L) or C_(H) from a different class ofimmunoglobulin molecule. This can be achieved using a vector or nucleicacid molecule that comprises a C_(L) or C_(H) chain, as known in theart. For example, an antibody that specifically binds HMW-MAA that wasoriginally IgM may be class switched to an IgG. Class switching can beused to convert one IgG subclass to another, such as from IgG₁ to IgG₂.

Fully human monoclonal antibodies include human framework regions. Thehuman framework regions can include the framework regions disclosed inone or both of SEQ ID NO: 5 or SEQ ID NO: 6 (these sequences include CDRsequences as well as framework sequences). However, the frameworkregions can be from another source. Additional examples of frameworksequences that can be used include the amino acid framework sequences ofthe heavy and light chains disclosed in PCT Publication No. WO2006/074071 (see, for example, SEQ ID NOs: 1-16), which is hereinincorporated by reference.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant onHMW-MAA. These antibody fragments retain the ability to specificallybind with the antigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of M912.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

IV. Immunoconjugates for Use in Therapeutic and Diagnostic Moieties

The human monoclonal antibodies, or functional fragments thereof, thatspecifically bind human HMW-MAA can be used in therapeutic methods. Inseveral embodiments, the human monoclonal antibodies or functionalfragments thereof described herein can be conjugated to a therapeuticagent. Immunoconjugates include, but are not limited to, molecules inwhich there is a covalent linkage of a therapeutic agent to an antibody.A therapeutic agent is an agent with a particular biological activitydirected against a particular target molecule or a cell bearing a targetmolecule. One of skill in the art will appreciate that therapeuticagents can include various drugs such as vinblastine, daunomycin and thelike, cytotoxins such as native or modified Pseudomonas exotoxin orDiphtheria toxin, encapsulating agents (such as liposomes) whichthemselves contain pharmacological compositions, radioactive agents suchas ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties andligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell, and the desired biological effect. Thus, forexample, the therapeutic agent can be a cytotoxin that is used to bringabout the death of a particular target cell. Conversely, where it isdesired to invoke a non-lethal biological response, the therapeuticagent can be conjugated to a non-lethal pharmacological agent or aliposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies described herein, one ofskill can readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same EM or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusion proteins thereof.

Effector molecules can be linked to an antibody of interest using anynumber of means known to those of skill in the art. Both covalent andnoncovalent attachment means may be used. The procedure for attaching aneffector molecule to an antibody varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule. Alternatively, the antibody is derivatized to exposeor attach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The antibodies or antibody fragments that specifically bind HMW-MAAdisclosed herein can be derivatized or linked to another molecule (suchas another peptide or protein). In general, the antibodies or portionthereof is derivatized such that the binding to HMW-MAA is not affectedadversely by the derivatization or labeling. For example, the antibodycan be functionally linked (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (for example, a bispecific antibodyor a diabody), a detection agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate associate of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

One type of derivatized antibody is produced by cross-linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

A human antibody that specifically binds HMW-MAA or functional fragmentthereof can be labeled with a detectable moiety. Useful detection agentsinclude fluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors and the like.Bioluminescent markers are also of use, such as luciferase, Greenfluorescent protein (GFP), Yellow fluorescent protein (YFP). An antibodycan also be labeled with enzymes that are useful for detection, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it can be detected by adding additionalreagents that the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase is presentthe addition of hydrogen peroxide and diaminobenzidine leads to acolored reaction product, which is visually detectable. An antibody mayalso be labeled with biotin, and detected through indirect measurementof avidin or streptavidin binding. It should be noted that the avidinitself can be labeled with an enzyme or a fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect HMW-MAA by x-ray,emission spectra, magnetic resonance imaging (MRI), commuted tomography(CT) scan, positron emission tomography (PET), or other diagnostictechniques. Examples of labels for polypeptides include, but are notlimited to, the following radioisotopes or radionucleotides: ³⁵F, ¹¹C,¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc, ¹³¹I, ³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, and¹²⁵I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Toxins can be employed with the HMW-MAA-specific human monoclonalantibodies, and functional fragments thereof, that are described herein,to produce immunotoxins. Exemplary toxins include ricin, abrin,diphtheria toxin and subunits thereof, as well as botulinum toxins Athrough F. These toxins are readily available from commercial sources(for example, Sigma Chemical Company, St. Louis, Mo.). Contemplatedtoxins also include variants of the toxins described herein (see, forexample, see, U.S. Pat. Nos. 5,079,163 and 4,689,401). In oneembodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No.5,602,095). As used herein “Pseudomonas exotoxin” refers to afull-length native (naturally occurring) PE or a PE that has beenmodified. Such modifications can include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib, IIand III, single amino acid substitutions and the addition of one or moresequences at the carboxyl terminus (for example, see Siegall et al., J.Biol. Chem. 264:14256-14261, 1989). In one embodiment, the cytotoxicfragment of PE retains at least 50%, at least 75%, at least 90%, or atlest 95% of the cytotoxicity of native PE. In some examples, thecytotoxic fragment is more toxic than native PE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The method of PEaction is inactivation of the ADP-ribosylation of elongation factor 2(EF-2). The exotoxin contains three structural domains that act inconcert to cause cytotoxicity. Domain la mediates cell binding. DomainII is responsible for translocation into the cytosol and domain IIImediates ADP ribosylation of elongation factor 2. The function of domainIb is unknown. PE employed with the monoclonal antibodies describedherein can include the native sequence, cytotoxic fragments of thenative sequence, and conservatively modified variants of native PE andits cytotoxic fragments. Cytotoxic fragments of PE include those whichare cytotoxic with or without subsequent proteolytic or other processingin the target cell. Cytotoxic fragments of PE include PE40, PE38, andPE35. For additional description of PE and variants thereof, see forexample, U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039;5,821,238; and 5,854,044; PCT Publication No. WO 99/51643; Pai et al.,Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol.Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta1333:C1-C6, 1997, each of which is herein incorporated by reference.

The antibodies and functional fragments thereof described herein canalso be used to target any number of different diagnostic or therapeuticcompounds to cells expressing HMW-MAA on their surface. Thus, anantibody of the present disclosure can be attached directly or via alinker to a drug that is to be delivered directly to cells expressingcell-surface HMW-MAA. Therapeutic agents include such compounds asnucleic acids, proteins, peptides, amino acids or derivatives,glycoproteins, radioisotopes, lipids, carbohydrates, or recombinantviruses. Nucleic acid therapeutic and diagnostic moieties includeantisense nucleic acids, derivatized oligonucleotides for covalentcross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-HMW-MAA antibody can be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (for example, anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art (see, for example, U.S. Pat. No. 4,957,735;Connor et al., Pharm. Ther. 28:341-365, 1985).

Antibodies described herein can also be covalently or non-covalentlylinked to a detectable label. Detectable labels suitable for such useinclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels include magnetic beads, fluorescent dyes (for example,fluorescein isothiocyanate, Texas red, rhodamine, green fluorescentprotein, and the like), radiolabels (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (such as horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic (such as polystyrene,polypropylene, latex, and the like) beads. These antibodies can be usedin a variety of immunoassays, including Fluorescence activated cellssorting (FACS), immunohistochemistry, radioimmune assays (RIAs), andenzyme-linked immunosorbant assays (ELISA).

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

V. HMW-MAA Antibody Polynucleotides and Polypeptides

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies, functional fragments thereof, immunoconjugates and fusionproteins) can readily be produced by one of skill in the art, using theamino acid sequences provided herein, sequences available in the art,and the genetic code. In addition, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which differ in sequence but which encode the sameeffector molecule or antibody sequence. Thus, nucleic acids encodingantibodies, conjugates and fusion proteins are provided herein.

In some embodiments, the HMW-MAA human monoclonal antibodies have aV_(H) domain encoded by a nucleotide sequence comprising SEQ ID NO: 1.In some embodiments, the HMW-MAA human monoclonal antibodies have aV_(L) domain encoded by the nucleotide sequence comprising SEQ ID NO: 3.In some embodiments, the HMW-MAA human monoclonal antibodies have aheavy chain comprising the nucleotide sequence of SEQ ID NO: 4.

Nucleic acid sequences encoding the human antibodies that specificallybind HMW-MAA, or functional fragments thereof that specifically bindHMW-MAA, can be prepared by any suitable method including, for example,cloning of appropriate sequences or by direct chemical synthesis bymethods such as the phosphotriester method of Narang et al., Meth.Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al.,Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phasephosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding human antibodies that specifically bindHMW-MAA, or functional fragments thereof that specifically bind HMW-MAA,can be prepared by cloning techniques. Examples of appropriate cloningand sequencing techniques, and instructions sufficient to direct personsof skill through many cloning exercises are found in Sambrook et al.,supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids encoding native effector molecule (EM) or anti-HMW-MAAantibodies can be modified to form the EM, antibodies, orimmunoconjugates of the present disclosure. Modification bysite-directed mutagenesis is well known in the art. Nucleic acids canalso be prepared by amplification methods. Amplification methods includepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (3SR). A wide variety of cloning methods,host cells, and in vitro amplification methodologies are well known topersons of skill.

In one embodiment, immunoconjugates are prepared by inserting the cDNAwhich encodes a human HMW-MAA-specific monoclonal antibody or functionalfragment thereof into a vector which comprises the cDNA encoding the EM.The insertion is made so that the antibody and the EM are read in frame,that is in one continuous polypeptide which contains a functionalantibody region and a functional EM region. In one embodiment, cDNAencoding an EM, label or enzyme is ligated to an antibody so that theEM, label or enzyme is located at the carboxyl terminus of the antibody.In another embodiment, the EM, label or enzyme is located at the aminoterminus of the antibody. In a another example, cDNA encoding the EM,label or enzyme is ligated to a heavy chain variable region of anantibody, so that the EM, label or enzyme is located at the carboxylterminus of the heavy chain variable region. The heavy chain-variableregion can subsequently be ligated to a light chain variable region ofthe antibody using disulfide bonds. In a yet another example, cDNAencoding an EM, label or enzyme is ligated to a light chain variableregion of an antibody, so that the EM, label or enzyme is located at thecarboxyl terminus of the light chain variable region. The lightchain-variable region can subsequently be ligated to a heavy chainvariable region of the antibody using disulfide bonds.

Once the nucleic acids encoding an EM, anti-HMW-MAA antibody, functionalfragment thereof, or an immunoconjugate, are isolated and cloned, thedesired protein can be expressed in a recombinantly engineered cell suchas bacteria, plant, yeast, insect and mammalian cells. It is expectedthat those of skill in the art are knowledgeable in the numerousexpression systems available for expression of proteins including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the isolated antibodies andantibody fragments described herein can be achieved by operably linkingthe DNA or cDNA to a promoter (which is either constitutive orinducible), followed by incorporation into an expression cassette. Thecassettes can be suitable for replication and integration in eitherprokaryotes or eukaryotes. Typical expression cassettes contain specificsequences useful for regulation of the expression of the DNA encodingthe protein. For example, the expression cassettes can includeappropriate promoters, enhancers, transcription and translationterminators, initiation sequences, a start codon (i.e., ATG) in front ofa protein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons.

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.For E. coli, this includes a promoter such as the T7, trp, lac, orlambda promoters, a ribosome binding site, and preferably atranscription termination signal. For eukaryotic cells, the controlsequences can include a promoter and/or an enhancer derived from, forexample, an immunoglobulin gene, SV40 or cytomegalovirus, and apolyadenylation sequence, and can further include splice donor andacceptor sequences. The cassettes can be transferred into the chosenhost cell by well-known methods such as transformation orelectroporation for E. coli and calcium phosphate treatment,electroporation or lipofection for mammalian cells. Cells transformed bythe cassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein (i.e., a human HMW-MAA-specific monoclonal antibody oran immunoconjugate comprising the antibody) without diminishing itsbiological activity. Some modifications can be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, termination codons, a methionine addedat the amino terminus to provide an initiation, site, additional aminoacids placed on either terminus to create conveniently locatedrestriction sites, or additional amino acids (such as poly His) to aidin purification steps. In addition to recombinant methods, theimmunoconjugates, effector moieties, and antibodies of the presentdisclosure can also be constructed in whole or in part using standardpeptide synthesis well known in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

VI. Compositions and Therapeutic Methods

Compositions are provided herein that include a carrier and one or moreof the antibodies that specifically bind HMW-MAA, or functional fragmentthereof that specifically binds HMW-MAA. Compositions comprisingimmunoconjugates or immunotoxins are also provided. The compositions canbe prepared in unit dosage forms for administration to a subject. Theamount and timing of administration are at the discretion of thetreating physician to achieve the desired purposes. The antibody can beformulated for systemic or local (such as intra-tumor) administration.In one example, the antibody that specifically binds HMW-MAA isformulated for parenteral administration, such as intravenousadministration.

The compositions for administration can include a solution of theantibody that specifically binds HMW-MAA (or a functional fragmentthereof) dissolved in a pharmaceutically acceptable carrier, such as anaqueous carrier. A variety of aqueous carriers can be used, for example,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day may be used,particularly if the agent is administered to a secluded site and notinto the circulatory or lymph system, such as into a body cavity or intoa lumen of an organ. Actual methods for preparing administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa.(1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In oneexample, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

The antibody that specifically binds HMW-MAA (or functional fragmentthereof) can be administered to slow or inhibit the growth of cells,such as cancer cells. In these applications, a therapeutically effectiveamount of an antibody is administered to a subject in an amountsufficient to inhibit growth, replication or metastasis of cancer cells,or to inhibit a sign or a symptom of the cancer. In some embodiments,the antibodies are administered to a subject to inhibit or prevent thedevelopment of metastasis, or to decrease the size or number ofmetasases, such as micrometastases, for example micrometastases to theregional lymph nodes (Goto et al., Clin. Cancer Res. 14(11):3401-3407,2008).

Suitable subjects may include those diagnosed with a cancer thatexpresses HMW-MAA, such as, but not limited to, melanoma, prostatecancer, squamous cell carcinoma (such as head and neck squamous cellcarcinoma), breast cancer (including, but not limited to basal breastcarcinoma, ductal carcinoma and lobular breast carcinoma), leukemia(such as acute myelogenous leukemia and 11q23-positive acute leukemia),a neural crest tumor (such as an astrocytoma, glioma or neuroblastoma),ovarian cancer, colon cancer, stomach cancer, pancreatic cancer, bonecancer (such as a chordoma), glioma or a sarcoma (such aschondrosarcoma).

A therapeutically effective amount of a human HMW-MAA-specific antibodywill depend upon the severity of the disease and the general state ofthe patient's health. A therapeutically effective amount of the antibodyis that which provides either subjective relief of a symptom(s) or anobjectively identifiable improvement as noted by the clinician or otherqualified observer. These compositions can be administered inconjunction with another chemotherapeutic agent, either simultaneouslyor sequentially.

Many chemotherapeutic agents are presently known in the art. In oneembodiment, the chemotherapeutic agents is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with acompound of the invention. Examples of useful COX-II inhibitors includeCELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples of usefulmatrix metalloproteinase inhibitors are described in PCT Publication No.WO 96/33172 (published Oct. 24, 1996), PCT Publication No. WO 96/27583(published Mar. 7, 1996), European Patent Application No. 97304971.1(filed Jul. 8, 1997), European Patent Application No. 99308617.2 (filedOct. 29, 1999), PCT Publication No. WO 98/07697 (published Feb. 26,1998), PCT Publication No WO 98/03516 (published Jan. 29, 1998), PCTPublication No WO 98/34918 (published Aug. 13, 1998), PCT Publication NoWO 98/34915 (published Aug. 13, 1998), PCT Publication No WO 98/33768(published Aug. 6, 1998), PCT Publication No WO 98/30566 (published Jul.16, 1998), European Patent Publication 606,046 (published Jul. 13,1994), European Patent Publication 931,788 (published Jul. 28, 1999),PCT Publication No WO 90/05719 (published May 31, 1990), PCT PublicationNo WO 99/52910 (published Oct. 21, 1999), PCT Publication No WO 99/52889(published Oct. 21, 1999), PCT Publication No WO 99/29667 (publishedJun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filedJul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar.25, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No.5,861,510 (issued Jan. 19, 1999), and European Patent Publication780,386 (published Jun. 25, 1997). In one example, the MMP inhibitors donot induce arthralgia upon administration. In another example, the MMPinhibitor selectively inhibits MMP-2 and/or MMP-9 relative to the othermatrix-metalloproteinases (such as MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specificexamples of MMP inhibitors of use are AG-3340, RO 32-3555, RS 13-0830,3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxaicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

The antibodies that specifically bind HMW-MAA can also be used withsignal transduction inhibitors, such as agents that can inhibit EGF-R(epidermal growth factor receptor) responses, such as EGF-R antibodies,EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascularendothelial growth factor) inhibitors, such as VEGF receptors andmolecules that can inhibit VEGF; and erbB2 receptor inhibitors, such asorganic molecules or antibodies that bind to the erbB2 receptor, forexample, HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are describedin, for example in PCT Publication Nos. WO 95/19970 (published Jul. 27,1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan.22, 1998), and U.S. Pat. No. 5,747,498 (issued May 5, 1998).EGFR-inhibiting agents also include, but are not limited to, themonoclonal antibodies C225 and anti-EGFR 22Mab (ImClone SystemsIncorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA),EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck KgaA), andthe compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701(Cephalon), leflunomide (Pharmacia/Sugen), Cl-1033 (Warner Lambert ParkeDavis), Cl-1033/PD 183,805 (Warner Lambert Parke Davis), CL-387,785(Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A(Bristol Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (BoehringerIngelheim), OLX-103 (Merck & Co.), VRCTC-310 (Ventech Research), EGFfusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808(Imperial Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (ParkerHughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974(Glaxo), KT-8391 (Kyowa Hakko) and EGF-R Vaccine (York Medical/Centro deImmunologia Molecular (CIM)).

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be used in conjunction withan antibody that specifically binds HMW-MAA. VEGF inhibitors aredescribed in, for example in PCT Publication No. WO 99/24440 (publishedMay 20, 1999), PCT International Application PCT/IB99/00797 (filed May3, 1999), PCT Publication No. WO 95/21613 (published Aug. 17, 1995), PCTPublication No. WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No.5,834,504 (issued Nov. 10, 1998), PCT Publication No. WO 98/50356(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat. No.5,792,783 (issued Aug. 11, 1998), PCT Publication No. WO 99/10349(published Mar. 4, 1999), PCT Publication No. WO 97/32856 (publishedSep. 12, 1997), PCT Publication No. WO 97/22596 (published Jun. 26,1997), PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCTPublication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and PCT Publication No. WO 98/02437 (publishedJan. 22, 1998). Other examples of some specific VEGF inhibitors areIM862 (Cytran Inc.); anti-VEGF monoclonal antibody of Genentech, Inc.;and angiozyme, a synthetic ribozyme from Ribozyme and Chiron. These andother VEGF inhibitors can be used in conjunction with an antibody thatspecifically binds HMW-MAA.

ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome pic), andthe monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), can furthermore be combined with the compound of theinvention, for example those indicated in PCT Publication No. WO98/02434 (published Jan. 22, 1998), PCT Publication No. WO 99/35146(published Jul. 15, 1999), PCT Publication No. WO 99/35132 (publishedJul. 15, 1999), PCT Publication No. WO 98/02437 (published Jan. 22,1998), PCT Publication No. WO 97/13760 (published Apr. 17, 1997), PCTPublication No. WO 95/19970 (published Jul. 27, 1995), U.S. Pat. No.5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305 (issuedMar. 2, 1999). ErbB2 receptor inhibitors of use are also described inU.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, and inU.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999.

For the treatment of cancer, such as melanoma, the antibodies disclosedherein can be used with surgical treatment, or with another therapeuticincluding dacarbazine (also termed DTIC), or interleukin-2 (IL-2) orinterferon, such as interferon (IFN). For the treatment of a superficialmelanoma, the antibodies can be used in conjunction with Imiquimod. Fortreatment of prostate cancer, the antibodies can be used in conjunctionwith, for example, surgery, radiation therapy, chemotherapy and hormonaltherapy (such as anti-androgens or GnRH antagonists). For the treatmentof HNSCC, the antibodies provided herein can be used in conjunction withsurgery, radiation therapy, chemotherapy, other antibodies (such ascetuximab and bevacizumab) or small-molecule therapeutics (such aserlotinib).

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies (or functional fragmentsthereof) disclosed herein to effectively treat the patient. The dosagecan be administered once but may be applied periodically until either atherapeutic result is achieved or until side effects warrantdiscontinuation of therapy. In one example, a dose of the antibody isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995) incorporated herein by reference.Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein, such as a cytotoxin or a drug, as a central core.In microspheres the therapeutic is dispersed throughout the particle.Particles, microspheres, and microcapsules smaller than about 1 μm aregenerally referred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

Fully human monoclonal antibodies that specifically bind HMW-MAA, or afunctional fragment thereof, covalently linked to an effector moleculecan be used for a variety of purposes, including for radioimmunotherapyor radioimmunoguided surgery. For example, a HMW-MAA antibody can belinked to a radioactive isotope and used in immunotherapy to treat atumor expressing HMW-MAA. A human HMW-MAA antibody covalently linked toa radioactive isotope is of use to localize a tumor in radioimmunoguidedsurgery, such that the tumor can be surgically removed. In oneembodiment, about 10 mCi of a radiolabeled human HMW-MAA monoclonalantibody is administered to a subject. In other embodiments, about 15mCi, about 20 mCi, about 50 mCi, about 75 mCi or about 100 mCi of aradiolabeled human HMW-MAA monoclonal antibody is administered to asubject. In other embodiments, about 100 mCi to about 100 mCi of aradiolabled human HMW-MAA monoclonal antibody is administered to asubject.

A method of detecting tumors in a subject includes the administration ofa human antibody that specifically binds HMW-MAA, or functional fragmentthereof, complexed to an effector molecule, such as a radioactiveisotope. After a sufficient amount of time has elapsed to allow for theadministered radiolabeled antibody to localize to the tumor, the tumoris detected. In one specific, non-limiting example, a radiolabeledimmune complex is detected using a hand held gamma detection probe. Insome embodiments, the tumor is detected by MRI, CT scan or PET scan.Primary tumors, metastasized tumors, or cells expressing HMW-MAA can bedetected. For example, a human HMW-MAA monoclonal antibody complexed toan effector molecule, such as a radioactive isotope, is administered toa subject prior to surgery or treatment. In one specific embodiment, thedetection step is performed prior to surgery to localize the tumor. Inanother embodiment, the detection step is performed during surgery, forexample to detect the location of the tumor prior to removing it, as inradioimmunoguided surgery. A human HMW-MAA monoclonal antibody complexedto an effector molecule, such as a radioactive isotope, can also beadministered to a subject following surgery or treatment, to determinethe effectiveness of the treatment, such as to ensure the completeremoval of the tumor, or to detect a recurrence of the tumor. Thus, theantibodies are of use as therapeutic agents (such as for immunotherapyagainst tumors) or for carrying out radioimmunoguided surgery.

VI. Diagnostic Methods and Kits

A method is provided herein for the detection of the expression ofHMW-MAA in vitro or in vivo. In one example, expression of HMW-MAA isdetected in a biological sample. The sample can be any sample,including, but not limited to, tissue from biopsies, autopsies andpathology specimens. Biological samples also include sections oftissues, for example, frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum,plasma, sputum, spinal fluid or urine.

In several embodiments, a method is provided for detecting a malignancysuch as melanoma, prostate cancer, squamous cell carcinoma (such as headand neck squamous cell carcinoma), breast cancer (including, but notlimited to basal breast carcinoma, ductal carcinoma and lobular breastcarcinoma), leukemia (such as acute myelogenous leukemia and11q23-positive acute leukemia), a neural crest tumor (such as anastrocytoma, glioma or neuroblastoma), ovarian cancer, colon cancer,stomach cancer, pancreatic cancer, bone cancer (such as a chordoma),glioma, or a sarcoma (such as chondrosarcoma). Serum samples frompatients with HMW-MAA-positive cancers contain detectable amounts ofHMW-MAA (Vergilis et al., J. Invest. Dermatol. 125:526-531, 2005; Ulmeret al., Clin. Cancer Res. 10:531-537, 2004). Thus, antibodies thatspecifically bind HMW-MAA, or functional fragments thereof, can be usedto detect HMW-MAA in a serum sample from a subject to detect cancer inthe subject, or confirm a diagnosis of cancer in a subject.

The disclosure provides a method for detecting HMW-MAA in a biologicalsample, wherein the method includes contacting a biological sample witha human antibody that binds HMW-MAA, or a functional fragment thereof,under conditions conducive to the formation of an immune complex, anddetecting the immune complex, to detect the HMW-MAA in the biologicalsample. In one example, the detection of HMW-MAA in the sample indicatesthat the subject has a malignancy. In another example, detection ofHMW-MAA in the sample confirms a diagnosis of cancer in a subject. In afurther example, detection of HMW-MAA confirms or detects the presenceof metastases.

In some embodiments, the fully human monoclonal antibody thatspecifically binds HMAW-MAA, or functional fragment thereof, is used fordetection or diagnosis of a tumor in a subject, such as confirming thediagnosis of a tumor in a subject. In other embodiments, the fully humanmonoclonal antibody that specifically binds HMAW-MAA, or functionalfragment thereof, is used to detect the efficacy of a therapy. Forexample, a subject with a known malignancy that expresses HMW-MAA isadministered a therapeutic agent. The method can include contacting abiological sample with a human antibody that binds HMW-MAA, or afunctional fragment thereof, under conditions conducive to the formationof an immune complex, and detecting the immune complex, to detect theHMW-MAA in the biological sample. A decrease in the amount of HMW-MAA,as compared to a control, such as a sample from the subject prior totreatment or a reference standard, indicates that the therapeutic agentis effective at treating the malignancy. In some examples, an increasein the amount of HMW-MAA, as compared to the control indicates that thetherapeutic agent is not effective for treating the malignancy.

In some embodiments, the detection can be in vivo. The human monoclonalantibody that specifically binds HMAW-MAA, or functional fragmentthereof, can be complexed to a radioactive isotope. After a sufficientamount of time has elapsed to allow for the administered radiolabeledantibody to localize to the tumor, the tumor is detected, such as byMRI, CT scan or PET scan.

In one embodiment, the human antibody that specifically binds HMW-MAA orfunctional fragment thereof is directly labeled with a detectable label.In another embodiment, the human antibody that specifically bindsHMW-MAA or functional fragment thereof (the first antibody) is unlabeledand a second antibody or other molecule that can bind the human antibodythat specifically binds HMW-MAA is labeled. As is well known to one ofskill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ³⁵S, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ¹⁹F, ^(99m)Tc, ¹³¹I,³H, ¹⁴C, ¹⁵N, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In and ¹²⁵I.

In an alternative embodiment, HMW-MAA can be assayed in a biologicalsample by a competition immunoassay utilizing HMW-MAA standards labeledwith a detectable substance and an unlabeled human antibody thatspecifically binds HMW-MAA. In this assay, the biological sample, thelabeled HMW-MAA standards and the human antibody that specifically bindHMW-MAA or functional fragment thereof are combined and the amount oflabeled HMW-MAA standard bound to the unlabeled antibody is determined.The amount of HMW-MAA in the biological sample is inversely proportionalto the amount of labeled HMW-MAA standard bound to the antibody thatspecifically binds HMW-MAA, or functional fragment thereof.

The immunoassays and method disclosed herein can be used for a number ofpurposes. In one embodiment, the human antibody that specifically bindsHMW-MAA or functional fragment thereof may be used to detect theproduction of HMW-MAA in cells in cell culture. In another embodiment,the antibody can be used to detect the amount of HMW-MAA in a biologicalsample. Increased expression of HMW-MAA is associated with several typesof cancer, including, but not limited to melanoma, breast cancer,prostate cancer, glioma and squamous cell carcinoma. In one embodiment,a kit is provided for detecting HMW-MAA in a biological sample, such asa serum sample or tissue sample. For example, to confirm a cancerdiagnosis in a subject, a biopsy can be performed to obtain a tissuesample for histological examination. Alternatively, a serum sample canbe obtained to detect the presence of HMW-MAA protein. Kits fordetecting a polypeptide will typically comprise a human antibody thatspecifically binds HMW-MAA, such as any of the antibodies disclosedherein. In some embodiments, an antibody fragment, such as an Fvfragment or scFv, or a Fab is included in the kit. In a furtherembodiment, the antibody is labeled (for example, with a fluorescent,radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds HMW-MAA. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting HMW-MAA in a biological samplegenerally includes the steps of contacting the biological sample with anantibody or antibody fragment which specifically reacts, underimmunologically reactive conditions, to a HMW-MAA polypeptide. Theantibody is allowed to specifically bind under immunologically reactiveconditions to form an immune complex, and the presence of the immunecomplex (bound antibody) is detected directly or indirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), enzyme linked immunosorbent assays (ELISA), orimmunohistochemical assays. The antibodies can also be used forfluorescence microscopy or fluorescence activated cell sorting (FACS). AFACS employs a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells (seeU.S. Pat. No. 5,061,620). Any of the human antibodies that specificallybind HMW-MAA, as disclosed herein, can be used in these assays. Thus,the antibodies can be used in a conventional immunoassay, including,without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry,Western blot or immunoprecipitation.

VII. Peptide Mimics

Also provided herein are peptides mimics of HMW-MAA. The peptide mimicsspecifically bind a human monoclonal antibody specific for HMW-MAA. Thepeptide mimics were identified by panning a peptide library withHMW-MAA-specific human monoclonal antibody scFv C21. The HMW-MAA peptidemimics can be used, for example, to elicit a HMW-MAA-specific immuneresponse in a subject diagnosed with a HMW-MAA-positive cancer, such asmelanoma.

Provided herein are isolated peptides that bind a human monoclonalantibody specific for HMW-MAA, wherein the peptide comprises theconsensus motif PXXYXPXXD (SEQ ID NO: 9). The peptide mimics aregenerally about 10 to about 20 amino acids in length, such as about 13to about 17, or about 15 amino acids in length. In some embodiments, thepeptides are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids inlength. In some examples, the amino acid sequence of the peptidecomprises SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or a variant ofSEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13, wherein the variantcomprises no more than three, no more than two or no more than one aminoacid substitutions. In particular examples, the amino acid sequence ofthe peptide consists of SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13.

Also provided is a method of inducing HMW-MAA-specific immunity in asubject, comprising administering to the subject the peptide comprisingthe consensus motif PXXYXPXXD (SEQ ID NO: 9).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Materials and Methods

Cell Lines, Cell Lysate and Tissues

The human melanoma cell lines Colo38, FO-1 and Melur and the human Blymphoid cell line LG2 were maintained in RPMI 1640 medium (TissueCulture Media Facility, Roswell Park Cancer Institute (RPCI), Buffalo,N.Y.) supplemented with 10% serum plus supplement (BioWhittaker,Walkersville, Md.) and 2 mM L-glutamine (BioWhittaker). The humanmelanoma cell lines M14 and SK-MEL-28, the human fibroblasts FF2376, thehuman breast carcinoma cell line T47D, the human bladder carcinoma cellline T24, the human prostate carcinoma cell line PC3, the human Blymphoid cell lines JY and LKT13, and the rat neural cell line B49 weremaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS) (BioWhittaker) and 2 mM L-glutamine. The M14/HMW-MAA cells whichexpress HMW-MAA following transfection of M14 cells with a plasmid DNAof pcDNA 3.1™ (+)/full length of HMW-MAA DNA construct (Yang et al., JCell Biol 165:881-891, 2004) were grown in RPMI 1640 medium supplementedwith 10% FBS, 2 mM L-glutamine and 0.4 mg/ml G418 (Promega, Madison,Wis.). Cells were cultured at 37° C. in a 5% CO₂ atmosphere. Celllysates were prepared as described (Desai et al., Cancer Res58:2417-2425, 1998, herein incorporated by reference). Lesions ofmelanocytic origin were obtained from patients who had undergone surgeryin the Department of Dermatology at Kumamoto University School ofMedicine (Kumamoto, Japan). The diagnosis of melanoma lesions was basedon histopathologic characteristics. The frozen and formalin-fixed tissuesections were prepared as described previously (Desai et al., Cancer Res58:2417-2425, 1998, herein incorporated by reference).

Animals

Eight-week old female BALB/c mice were obtained from the animal corefacility at RPCI.

Monoclonal and Polyclonal Antibodies, scFv Antibodies and Reagents

The HMW-MAA-specific mouse mAbs 149.53, 225.28, 763.74, TP61.5 andVF1-TP34 and VF1-TP41.2, were developed and characterized as describedelsewhere (Wilson et al., Int J Cancer, 28:293-300, 1981; Giacomini etal., Cancer Res 43:3586-3590, 1983; Chen et al., Cancer Res51:4790-4797, 1991; Temponi et al., Cancer Res 52:2497-2503, 1992).Cross-blocking experiments have shown that the 6 mAbs recognize distinctand spatially distant antigenic determinants, since they do notcross-inhibit each other in their binding to HMW-MAA⁺ melanoma cells(Campoli et al., Crit. Rev Immunol 24:267-296, 2004). The 100KD-specific mouse mAb 376.96 (Imai et al., J Natl Cancer Inst68:761-769, 1982), the HLA class I antigen-specific mouse mAb TP25.99(Desai et al., J Immunol 165:3275-3283, 2000), the c-myconcoprotein-specific mouse mAb 9E10 (Evan et al., Mol Cell Biol5:3610-3616, 1985) and the mouse anti-id mAb MK2-23 (Kusama et al., JImmunol 143:3844-3852, 1989) have been previously described. TheHMW-MAA-specific human scFv #28 (Noronha et al., J. Immunol.161:2968-2976, 1998), #61 (Desai et al., Cancer Res 58:2417-2425, 1998),and #70 (Noronha et al., J. Immunol. 161:2968-2976, 1998) and theanti-anti-id scFv #119 (Wang et al., Idiotypes in Medicine:Autoimmunity, Infection and Cancer p. 523, 1997) were isolated from thesynthetic scFv library (#1) (Nissim et al., Embo J 13:692-698, 1994) bypanning with melanoma cells S5, purified HMW-MAA, melanoma cellsSK-MEL-28 and anti-id mAb MK2-23, respectively. The MAA-specific humanscFv F98 and W34 were isolated from the semi-synthetic scFv library (deKruif et al., J Mol Biol 248:97-105, 1995) by panning with the melanomacells FO-1 and WM1158, respectively.

Mouse mAbs were purified from ascitic fluid by sequential ammoniumsulphate and caprylic acid precipitation (Temponi et al., Hybridoma8:85-95, 1989). The purity and activity of mAb preparations wereassessed by SDS-PAGE and by testing with the corresponding antigen in abinding assay, respectively. Monoclonal antibodies and purified scFvantibodies were biotinylated using NHS-LC-biotin (Pierce, Rockford,Ill.) according to the manufacturer's instructions. mAb 9E10 wasimmobilized on a HiTrap NHS-activated sepharose column (AmershamBiosciences, Piscataway, N.J.) following the manufacturer'sinstructions.

Streptavidin-horseradish peroxidase conjugate (SA-HRP) was purchasedfrom Pierce. HRP-anti-mouse IgG Fc antibodies were purchased fromJackson ImmunoResearch Laboratories, Inc., West Grove, Pa.R-phycoerythrin (RPE)-labeled F(ab′)₂ fragments of goat anti-mouse Igantibodies were purchased from BD Pharmingen, San Diego, Calif.

Phage Display Libraries

A large semi-synthetic phage display scFv antibody library with designedCDR3 was constructed as described (de Kruif et al., J Mol Biol248:97-105, 1995). Phage display peptide libraries X15 displaying 15amino acid, random linear peptides and LX-8 (XCX8CX) displaying 12 aminoacid, random, disulfide constrained peptides were constructed asdescribed (Bonnycastle et al., J Mol Biol 258:747-762, 1996).

Synthetic Peptides

Peptides were purchased from RPCI Biopolymer Core Facility (Buffalo,N.Y.). Peptide P1C21, containing cysteine residues, was cyclized with 6mM potassium ferricyanide and purified with reversed-phase HPLC. Thecomposition and disulfide bond formation were confirmed by massspectrometry. Purity of peptides was greater than 95%, as assessed byHPLC. Peptides were reconstituted in water at a concentration of 5 mM,aliquoted and stored at −20° C. Peptide P1C21, which is insoluble inwater, was reconstituted in dimethyl sulfoxide (DMSO) at a concentrationof 5 mM. Peptides were conjugated to keyhole limpet haemocyanin (KLH)(Pierce) using the coupling agent maleimidobenzoyl-N-hydroxysuccinimide(MBS) (Pierce) as described (Grant, Current Protocols in Immunology p.9.2.8, 2002).

Selection of Phage Display scFv Antibodies

Phage display scFv antibodies binding to melanoma cells were isolatedfrom the phage display scFv antibody library utilizing the panningtechnique as described (Noronha et al., J Immunol 161:2968-2976, 1998,herein incorporated by reference).

Preparation and Purification of Soluble scFv Antibodies

Soluble scFv antibodies were produced from individualampicillin-resistant E. Coli TG1 infected colonies by induction withisopropyl-β-D-thiogalactopyranoside (IPTG) (Roche Applied Science,Indianapolis, Ind.) as described (Schier et al., J Mol Biol 255:28-43,1996, herein incorporated by reference). Soluble scFv antibodies wereharvested from culture supernatants (SNT) or from the periplasmic space(periplasmic preparation, PP) of cultures of individual bacterialcolonies. scFv antibodies were purified from SNT or PP by affinitychromatography on insolubilized mAb 9E10 following the methodologydescribed for the RPAS purification module (Amersham). Purified scFvantibodies were concentrated using Centricon 10 (Millipore Corporation,Bedford, Mass.) following the manufacturer's instructions. The purityand activity of scFv antibody preparations was assessed by SDS-PAGEutilizing the PhastSystem™ (Amersham) and by testing with thecorresponding antigen utilizing enzyme linked immunosorbent assay(ELISA), respectively.

Binding Assays

The ELISA to test the reactivity of soluble scFv antibodies withsynthetic peptides and melanoma cells and of mouse immune sera withmelanoma cells and with peptides was performed as described (Desai etal., Cancer Res 58:2417-2425, 1998; Desai et al., J Immunol165:3275-3283, 2000; Matsui et al., J Immunol 139:2088-2095, 1987).Results are expressed as absorbance of optical density (O.D.) at 450 nmThe competition assay to map the antigenic determinant recognized byscFv C21 was performed by mixing biotinylated scFv C21 (0.25 μg/well)with two-fold dilutions of mouse mAb or scFv PP or by mixingbiotinylated mAb (at an optimal amount giving absorbance, measured at450 nm of 1.0) with two fold dilutions of scFv PP. The mixture (100μl/well) was incubated for 1 h at 4° C. with HMW-MAA⁺ cells (2×10⁵/50 μlof RPMI 1640 medium) in a 96-well tissue culture plate (Falcon 3072,Becton Dickinson, Franklin Lakes, N.J.). Binding of biotinylated scFvantibodies and biotinylated mAb to target cells was measured bysequential incubation with SA-HRP and substrate as described (Noronha etal., J Immunol 161:2968-2976, 1998). The results are expressed aspercent inhibition by mAb, scFv or peptide (competitor or inhibitor) ofscFv or mAb binding to HMW-MAA⁺ cells. The percent inhibition wascalculated using the formula: % inhibition=((OD₄₅₀ in the absence ofinhibitor−OD₄₅₀ in the presence of inhibitor)/OD₄₅₀ in the absence ofinhibitor)×100.

The indirect immunoperoxidase staining of frozen and formalin-fixedtissue sections was performed as described previously (Desai et al.,Cancer Res 58:2417-2425, 1998, herein incorporated by reference).

Indirect Immunoprecipitation and SDS-PAGE

Labeling of cells with ¹²⁵Iodine (Na¹²⁵I; Amersham) or with ³⁵Smethionine (Trans-³⁵S-label, ICN Biochemicals, Costa Mesa, Calif.) inthe presence of tunicamycin (Sigma Chemical Co.) was performed asdescribed (Noronha et al., J Immunol 161:2968-2976, 1998; Desai et al.,J Immunol 165:3275-3283, 2000). Solubilization of labelled cells,immunoprecipitation, SDS-PAGE, autoradiography and fluorography wereperformed as described (Desai et al., Cancer Res 58:2417-2425, 1998)except for the use of Gammon Bind plus sepharose (Amersham) instead ofprotein A coated with rabbit anti-mouse IgG antibodies.

Panning of Peptide Libraries with Biotinylated scFv C21

Micropanning of pVIII libraries X15 and LX-8 with biotinylated scFv C21was performed in 96 well microtiter plates (Falcon 3076, BectonDickinson) essentially as described (Desai et al., J Immunol165:3275-3283, 2000, herein incorporated by reference). The first roundof panning was performed with 1×10¹² phage particles in TBS 50 andbiotinylated scFv C21 at a concentration of 1 μg per well. Thesubsequent three rounds of panning were carried out utilizing a phageinput of 1×10¹⁰ phage particles and 0.1 μg per well of biotinylated scFvC21.

Immunological Screening of Phage Display Peptide Libraries

Random phage clones from X15 and LX-8 libraries after the fourth roundof panning with scFv C21 were analyzed by immunological screening asdescribed (Desai et al., J Immunol 165:3275-3283, 2000, hereinincorporated by reference) except for the use of 10 μg/ml of scFvantibody and 5 μg/ml of biotinylated mAb 9E10 to probe thenitrocellulose filter lifts from plates containing colonies.

Sequence Analysis of Phage Display Peptides

The sequence of peptide inserts of phage clones was determined by thedideoxynucleotide chain termination method, as described (Desai et al.,J Immunol 165:3275-3283, 2000).

Immunization of Mice

BALB/c mice (8 in each group) were immunized subcutaneously (s.c.) withP1C21 peptide-KLH conjugate (50 μg/injection) mixed with an equal volumeof complete Freund's adjuvant for priming on day 0 and of incompleteFreund's adjuvant for boosting on day 21, 42, 63, 84 and 105. Miceimmunized with the irrelevant peptide MB1₁₉₄₋₂₀₈ derived from thesequence of the proteasome subunit MB1 were used as controls. On day132, mice were boosted with a s.c. injection of irradiated (20K Rads)cells (5×10⁵ cells/mouse). Mice were bled one week before the firstimmunization and one week after each immunization.

Flow Cytometry Analysis

Flow cytometry analysis of melanoma cells stained with scFv antibodiesor antibodies in immune sera was performed as described (Wang et al., JImmunol Methods 294:23-35, 2004, herein incorporated by reference).Briefly, cells (5×10⁵) were incubated for 1 h at 4° C. with 12.5 μl ofscFv PP or with 0.5 μg of mAb 9E10 (both diluted in a total volume of100 μl of 2% BSA-PBS) or 100 μl of immune mouse sera. Cells were thenwashed twice with 0.5% BSA-PBS and incubated for 30 min at 4° C. with anoptimal amount of RPE-labeled F(ab′)₂ fragments of goat anti-mouse Igantibodies. Following two washes, cells were fixed in 2% formaldehydeand analyzed with a FACScan™ flow cytometer (BD Biosciences, San Jose,Calif.). A total of 10,000 cells were counted using a forward and sidescatter gate to eliminate aggregates and debris for each sample. Resultsare expressed as relative fluorescence intensity.

Delayed-Type Hypersensitivity (DTH) Reaction

Mice which had been immunized six times with peptide P1C21 or controlpeptide were injected on day 132 s.c. into the right and left hindfootpads with irradiated HMW-MAA⁺ cells Colo38 (5×10⁵ cells/injection/)and HMW-MAA⁻ cells LG2 (5×10⁵ cells/injection/), respectively. Thethickness of each footpad was measured and calculated at the indicatedtimes as previously described (Luo et al., J Immunol 174:7104-7110,2005, herein incorporated by reference).

Statistical Analysis

The statistical significance of the difference among the resultsobtained in the tested groups was analyzed using the Student's t-test.

Molecular Model of scFv C21-Peptide Complex

The molecular model of scFv C21 was built using AbM (Accelrys, SanDiego, Calif.). The VL-VH dimer was created using the crystal structureof influenza virus neuraminidase-(1NMC) (Tulip et al., J Mol Biol227:149-159, 1992) and lysozyme-(Ay et al., J Mol Biol 301:239-246,2000) specific scFv antibodies from protein database (Bernstein et al.,J Mol Biol 112:535-542, 1977). Classification and numbering schemes todefine CDR were according to Kabat et al (Kabat et al., In Sequences ofProteins of Immunological Interest 5:91, 1991). The CDR loops L1, H1, L2and H2 adopt standard canonical conformation. H3 and L3 were built usingboth loop search of protein database (INSIGHTII, Accelrys) and CONGEN(Bruccoleri et al., Nature 335:564-568, 1988). The startingconformations of peptides P1C21 and P3C21 were determined using loopsearch method built in INSIGHTII. Putative binding interactions ofpeptides P1C21 and P3C21 with scFv C21 were determined using combinationof software AUTODOCK (Morris et al., J Comput Aided Mol Des 10:293-304,1996), INSIGHTII and DOCK (DesJarlais et al., J Med Chem 29:2149-2153,1986).

Kinetic Binding Studies

Binding experiments were performed with the surface plasmon resonancebased biosensor instrument BIACORE™ 3000 (Biacore AB, Uppsala, Sweden),at 25° C. Immobilization of scFv C21 in the sensor surface was performedfollowing the standard amine coupling procedure according to themanufacturer's instructions. Briefly, 35 μl of a solution containing 0.2M N-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) and 0.05MN-hydroxysuccinimide (NHS), were injected at a flow rate of 5 μl/min toactivate carboxyl groups on the sensor chip surface. scFv C21 (40 ng/mlof 10 mM NaOAc buffer, pH 5.0) was flowed over the chip surface at aflow rate of 20 μl/min until the desired bound protein level wasreached. Unreacted scFv C21 was washed out and unreacted activatedgroups were blocked by the injection of 35 μl of 1 M ethanolamine at 5μl/min The final immobilization response of scFv C21 was 3,000 RU. Areference surface was generated simultaneously under the same conditionsbut without scFv C21 injection and used as a blank to correct forinstrument and buffer artifacts. Peptides were injected at variableconcentrations at different flow rates from 20-100 μl/min Peptidesbinding to scFv C21 immobilized on the chip was monitored in real time.To exclude that binding constants obtained from SPR were not affected byeither rebinding of the analyte or mass transport effects, studies wererepeated at different flow rates (20-100 μl/min). Kinetic constantsobtained at both flow rates remained unchanged.

Example 2 Isolation and Characterization of scFv C21

Isolation of scFv Antibodies by Panning the Semi-Synthetic Phage DisplayscFv Antibody Library with HMW-MAA⁺ Cells Colo38

Screening in ELISA with HMW-MAA⁺ cells Colo 38 and with HMW-MAA⁻ Blymphoid cells LG2 of 40 soluble scFv antibodies isolated from thesemi-synthetic phage display scFv antibody library by panning withColo38 cells identified 23 clones with selective reactivity with Colo38cells. Additional testing of the 23 clones with Colo38, FF2376fibroblasts and T24 bladder carcinoma cells resulted in the isolation ofthe clones C3, C21, C29 and C30, all of which displayed selectivereactivity with Colo38 cells. scFv C21 was selected for furtheranalysis, since it displayed the highest reactivity. The other clonescould not be characterized since they were lost.

Analysis of the Specificity of scFv C21

When tested in ELISA with a panel of human cell lines with differentialHMW-MAA expression, and with the rat neural cell line B49, whichexpresses a HMW-MAA homologue, scFv C21 reacted only with the melanomacell lines Colo38, FO-1, Melur and SK-MEL-28. All of them expressHMW-MAA, suggesting that scFv C21 is specific for HMW-MAA (FIG. 1). Thispossibility was proven by three lines of evidence. First, flow cytometryanalysis showed that scFv C21 stained M14 cells which express HMW-MAAfollowing stable transfection with a plasmid DNA encoding the fulllength HMW-MAA, but did not stain the parental M14 cells which do notexpress HMW-MAA (FIG. 2). Second, scFv C21 immunoprecipitated twocomponents with the characteristic elecrophoretic profile of HMW-MAAcomponents from ¹²⁵I labeled Colo38 cells. Lastly, in sequentialimmunoprecipitation experiments, scFv C21 did not immunoprecipitate anycomponents from a Colo38 cell lysate, which had been immunodepleted withthe HMW-MAA-specific mouse mAb 763.74. The immunodepletion is specific,since scFv C21 immunoprecipitated the HMW-MAA components from a Colo 38cell lysate immunodepleted with the 100 KD MAA-specific mAb 376.96 (FIG.3). Conversely, immunodepletion of a Colo38 cell lysate with scFv C21removed most, but not all the components immunoprecipitated by mAb763.74. These results reflect the expression of the determinantrecognized by scFv C21 on most, but not all the HMW-MAA moleculesrecognized by mAb 763.74 and/or the lower association constant of scFvC21 than that of mAb 763.74.

Characterization of the Antigenic Determinant Defined by scFv C21

Carbohydrates appear to play a role in the expression of the determinantdefined by scFv C21 because of the marked reduction in the intensity ofthe HMW-MAA components immunoprecipitated from Colo38 cells which hadbeen intrinsically labeled with ³⁵S-methionine in the presence of theN-glycosylation inhibitor tunicamycin. The inhibition of N-glycosylationis indicated by the accumulation of the 220 kD precursor which reactswith mAb 763.74 (FIG. 4). Competition experiments investigated thespatial relationship of the determinants defined by scFv C21 with the 6determinants defined by a panel of mouse mAb and with those defined byhuman scFv #28, #61 and #70. scFv C21 and mAb VF1-TP34 partiallyinhibited each other in their binding to melanoma cells SK-MEL-28. Theinhibition is dose dependent (FIG. 5). In contrast, the mouse mAb149.53, 225.28, 763.74, TP61.5 and VF1-TP41.2 and the scFv #28, #61 and#70 did not inhibit the binding of scFv C21 to SK-MEL-28 cells. Theseresults indicate that the determinant defined by scFv C21 is distinctand spatially close to that defined by mAb VF1-TP34 and is distinct andspatially distant from those defined by the remaining mouse mAb and bythe human scFv #28, #61 and #70.

Immunohistochemical Staining of Melanocytic Lesions by scFv C21

scFv C21 stained frozen melanocytic lesions in the immunoperoxidasereaction, but did not stain formalin fixed paraffin embedded melanocyticlesions. The staining was both membranous and cytoplasmic (FIG. 6).Comparison of the staining patterns of benign and malignant melanocyticlesions with scFv C21 and HMW-MAA-specific mouse mAb 763.74 showed thatboth antibodies stain homogeneously 4 pigmented nevi with a membranousand a cytoplasmic pattern. Furthermore, mAb 763.74 stained 14 out of 15primary lesions with a homogeneous pattern, while scFv C21 stained 9with a homogenous pattern and 2 with a heterogeneous pattern. Thestaining was membranous in 1 lesion, membranous and cytoplasmic in 4 andcytoplasmic in 6. Lastly, mAb 763.74 stained 6 metastatic lesions with ahomogeneous pattern, while scFv C21 stained 3 with a homogeneous patternand 3 with a heterogeneous pattern. The staining was membranous andcytoplasmic in 3 lesions and cytoplasmic in the remaining 3.

Example 3 Nucleotide and Amino Acid Sequences of scFv C21

The nucleotide and amino acid sequences of scFv C21 were determinedaccording to standard methods. The nucleotide and amino acid sequencesof the V_(H), V_(L) and linker are provided below. Also provided is thenucleotide sequence of the immunoglobulin heavy chain hinge, CH₂ and CH3domains.

Nucleotide Sequences

V_(H) (SEQ ID NO: 1) ATGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGCGATCTGACGACACGGCCGTGTATTACTGTGCAAGGGCCCTTGATCCTATTACGTTTGACTCCTGGGGCCAAGGTACCCTGGT CACCGTCTCGAGA Linker(SEQ ID NO: 2) GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG V_(L)(SEQ ID NO: 3) TCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTAGATCT Heavy Chain (Hc) Hinge + CH2 + CH3(SEQ ID NO: 4) GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCGGGCAAATGAAmino Acid Sequences

V_(H) (SEQ ID NO: 5) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCAR ALDPITFDSWGQGTLVTVSR(118 amino acids) V_(L) (SEQ ID NO: 6)SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVF GGGTKLTVLGRS(110 amino acids)

For both V_(H) (SEQ ID NO: 5) and V_(L) (SEQ ID NO: 6), the location ofthe framework regions (FR) and CDRs is as follows:

FR1—amino acids 1-26

CDR1—amino acids 27-38

FR2—amino acids 39-55

CDR2—amino acids 56-65

FR3—amino acids 66-104

CDR3—amino acids 105-115 (V_(H)) or 105-110 (V_(L))

Example 4 Identification and Characterization of scFv Peptide Mimics

Sequence of Peptides Binding to scFv C21 Isolated from Phage DisplayPeptide Libraries LX-8 and X15

Immunological screening revealed that scFv C21 reacted strongly with 20%of the clones isolated by panning from the phage display peptide X15library, but reacted with none of those isolated from the LX-8 library.The phage supernatants of positive clones obtained from immunoscreeningreacted with scFv C21 also in ELISA. The reactivity is specific, sincethe clones did not react with scFv F98 and W34 which recognize unrelatedMAA. Nucleotide sequence analysis of 16 of the clones reacting with scFvC21 identified the sequences SPSWYCPDCDKRPLV (P1C21) (SEQ ID NO: 7),EARNWHDFPIHPRTL (P2C21) (SEQ ID NO: 8) and RPYRYDPLGDLKSRH (P3C21) (SEQID NO: 10) in 88, 6 and 6%, respectively, of the 16 clones analyzed. Thesequences of peptides P1C21 and P3C21 share the consensus PXXYXPXXD (SEQID NO: 9), while that of peptide P2C21 is completely different.Consistent with the carbohydrate nature of the determinant recognized byscFv C21, no homology was found between the sequences of the threepeptides isolated from the phage display peptide libraries and thepublished amino acid sequence of the HMW-MAA core protein (Pluschke etal., Proc Natl Acad Sci USA 93:9710-9715, 1996).

Analysis of the Reactivity of Synthetic Peptides with scFv C21

To corroborate the reactivity of phage display peptides with scFv C21,peptides P1C21 (cyclized), P2C21 (linear), and P3C21 (linear), weresynthesized and tested for their ability to react in ELISA with scFv C21and to inhibit its binding to HMW-MAA⁺ melanoma cells Colo38 in aninhibition assay. The synthetic peptide P1C21 and P2C21, immobilized ona microtiter plate by 0.25% glutaraldehyde PBS, reacted specificallywith scFv C21 in ELISA while immobilized synthetic peptide P3C21 did not(FIG. 7). The binding is specific, since no peptides reacted with theunrelated HMW-MAA-specific scFv #28. Moreover, the peptides P1C21 andP3C21 inhibited the binding of scFv C21 to HMW-MAA⁺ cells in a dosedependent fashion (FIG. 8). The inhibition is specific since bothpeptides did not inhibit the binding of scFv #28 to melanoma cellsColo38 (FIG. 8). In contrast, P2C21 did not inhibit the binding of scFvC21 to melanoma cells Colo38. The inhibition of the binding of scFv C21to melanoma cells by soluble peptide P3C21 and the lack of reactivity ofimmobilized peptide P3C21 with scFv C21 suggest that the binding of thispeptide with scFv C21 is conformationally sensitive.

Induction of HMW-MAA-Specific Humoral and Cellular Immunity by PeptideP1C21 in BALB/c Mice

To further prove the HMW-MAA mimicry by peptide P1C21, its ability toelicit HMW-MAA-specific humoral and cellular immunity in BALB/c mice wastested. Antibodies selectively reacting with HMW-MAA⁺ melanoma cellsColo38 were detected in sera harvested from BALB/c mice one weekfollowing the third immunization with the peptide P1C21. The mean titerof these antibodies was low, although significantly (p<0.05) higher thanthat of the antibodies in sera from mice immunized with the unrelatedMB1₁₉₄₋₂₀₈ peptide. Furthermore, the titer of the antibodies reactingwith melanoma cells did not change following three additional boosters.However the titer of the antibodies selectively reacting with HMW-MAA⁺melanoma cells was markedly enhanced by boosting the mice immunized withpeptide P1C21 with HMW-MAA⁺ melanoma cells (FIG. 9).

Two lines of evidence proved the HMW-MAA specificity of the elicitedantibodies. First, the immune sera stained M14#5/HMW-MAA cells thatexpress HMW-MAA following transfection with a plasmid DNA encoding thefull length HMW-MAA, but did not stain the parental M14#5 cells (FIG.10). The latter cells do not express HMW-MAA. In contrast, sera frommice immunized with peptide MB1₁₉₄₋₂₀₈ displayed no reactivity withColo38 cells, although they highly reacted with the immunizing peptide.Second, SDS-PAGE analysis showed that sera from mice sequentiallyimmunized with peptide P1C21 and Colo38 cells immunoprecipitated thecharacteristic HMW-MAA components from a Colo38 cell lysate. Thespecificity of the immune response elicited by the prime-boost strategywith peptide P1C21 and HMW-MAA⁺ cells Colo38 is indicated by the lack ofselective reactivity with HMW-MAA⁺ cells Colo38 of sera from BALB/c miceimmunized (i) with peptide P1C21 and HMW-MAA⁻ lymphoid cells LG2 or (ii)with peptide MB1₁₉₄₋₂₀₈ and HMW-MAA⁺ cells Colo38, both in bindingassays and in immunoprecipitation experiments (FIG. 11).

Immunization of BALB/c mice with peptide P1C21 elicited a DTH responseto HMW-MAA⁺ cells Colo38 as indicated by the significantly (p<0.05)higher swelling of the footpad injected with Colo38 cells than of thatinjected with LG2 cells. The swelling persisted for 48 hours. The DTHresponse to HMW-MAA⁺ melanoma cells induced by peptide P1C21 isspecific, since no swelling of the footpad was observed in mice that hadbeen immunized with the unrelated MB1₁₉₄₋₂₀₈ peptide and challenged withColo38 cells (FIG. 12).

Structural Basis of Peptides P1C21 and P3C21 Binding to scFv C21

To define the structural basis for the binding of the peptides P1C21 andP3C21 to scFv C21, a molecular model of scFv C21 and its complex withthe two peptides was built. Starting models of the peptides P1C21 andP3C21 were built using the limited conformational search algorithm inINSIGHTII. The initial orientation of peptide binding to scFv C21 wasdetermined by DOCK analysis. The best orientation was selected based onthe contact score, as well as on the binding energy, and then minimizedusing INSIGHTII for optimal binding and interaction analysis (Table 1).During the minimization, scFv C21 was held fixed for the first 2000cycles. Subsequently, CDR loops and peptides were allowed to move.

TABLE 1 Intermolecular contacts of peptides P1C21 and P3C21 with scFvC21 and calculated binding energy of the respective complexes. TotalEnergy Ligand scFv C21 contacts (Kcal/mol) P1C21 (Ser2) H30, H31, H100−93.8 P1C21 (Pro 7) L92 P1C21 (Asp 8) L91, L30 P1C21 (Lys 11) H51, H57,H58 P1C21 (Arg 12) H32, H98, H99, H101 P1C21 (Pro 13) H53, H54 P1C21(Leu 14) H29 P3C21 (Pro 2) H28, H53 −7.3 P3C21 (Tyr 3) H30 P3C21 (Arg 4)H26, H27, H31 P3C21 (Tyr 5) H29 P3C21 (Pro 7) H99, H100 P3C21 (Leu 11)L30, L48 P3C21 (Lys 12) L50, L51 P3C21 (Arg 14) L29, L49, L64, L66 P3S10(Pro 2) H28, H53 −16.4 P3S10 (Tyr 3) H30 P3S10 (Arg 4) H26, H27, H31P3S10 (Tyr 5) H29 P3S10 (Pro 7) H99 P3S10 (Leu 8) H100 P3S10 (Ser 10)L47 P3S10 (Leu 11) L30, L48, L89 P3S10 (Lys 12) L50, L51 P3S10 (Arg 14)L29, L49, L64, L66

Molecular modeling of the binding mode conformations of the peptidesP1C21 and P3C21 indicates that the peptides can interact with differentfunctional groups within the antigen-combining site of scFv C21.Substitution of 10Ser for 10Asp is suggested to improve the binding ofthe linear peptide form. Residues shown in bold are extra favorablecontacts due to 10Asp to 10Ser mutation.

The primary sequences of the peptides P1C21 and P3C21 are 50%homologous. 5Tyr, 7Pro and 10Asp are conserved in both sequences at thecore. In the molecular modeling, both peptides interact with scFv C21through conserved residues, but in two orientations (Table 1 and FIG.13). The peptide P1C21 adopts a folded structure and binds across theheavy and light chain CDR3 of scFv C21, while the peptide P3C21 bindsacross all the CDR loops in an extended conformation. P1C21 is a cyclicpeptide and its folded structure makes several favorable interactionswith scFv C21 and also forms a more stable complex (Table 1), due to Trpat position 4 which docks into a hydrophobic pocket formed by heavychain CDR2 and CDR3 and by light chain CDR2. The linear peptide P3C21,on the other hand, forms a stable complex with fewer contacts throughTyr and mostly hydrophobic interactions mediated by Pro. The conserved10Asp is not involved in any contacts.

Effect of Amino Acid Substitutions on the Reactivity of Peptide P3C21with scFv C21

To further characterize effects associated with possible conformationaltransitions and potential contact residues, amino acid substitutionswere introduced into the linear peptide P3C21 based on the molecularmodeling studies. The effect of select substitutions on the reactivityof the peptide P3C21 with scFv C21 was tested experimentally by Biacore(Table 2) and inhibition analysis (FIG. 8).

TABLE 2 Kinetic binding analysis of peptide P1C21and P3C21 binding to scFv C21 Peptide K_(on) (1/Ms) K_(off) (1/s) Kd (M)P1C21 1.183e3 9.7e−3 8.24e−6 SPSWYCPDCDKRPLV (SEQ ID NO: 7) P3C21 2.17e36.55e−2 3.02e−5 RPYRYDPLGDLKSRH (SEQ ID NO: 10) P3A5 No detectableRPYRADPLGDLKSRH binding observed  (SEQ ID NO: 11) P3V7 No detectableRPYRYDVLGDLKSRH binding observed (SEQ ID NO: 12) P3S10 9.17e2 3.07e−33.34e−6 RPYRYDPLGSLKSRH (SEQ ID NO: 13) To evaluate binding, peptidesP1C21 and P3C21 were injected at concentrations ranging from 1 to 32 μM.The resulting sensograms were used for estimation of the kineticconstants (k_(on) and k_(off)) by global fitting to the 1:1 Langmurianinteraction model. The binding constant (K_(d)) was calculated for eachpeptide as a k_(off)/k_(on) ratio.

In the scFv C21-P3C21 complex, the interaction of 5Tyr (to H29) wascritical to keep the 7Pro anchored to the hydrophobic pocket, suggestingthat substitutions at these two critical positions would abrogate scFvC21 binding. Biacore analysis of the interactions between the peptidesand the immobilized scFv C21 was performed to test the effect of theintroduced modifications in the peptide sequences on their bindingproperties (Table 2). Good fitting of the experimental data to thecalculated curves was observed, suggesting a simple pseudo-first orderinteraction between the peptides and scFv C21. Substituted residues atpositions 5Tyr and 7Pro indeed abrogated peptide binding to scFv C21(Table 2).

In contrast to positions 5Tyr and 7Pro, 10Asp was not involved in anycontacts with scFv C21. However, during the simulation we noticed thatthe LGDL sequence tract was highly mobile, whereas 10Asp made frequentcontact (L89 Arg) with the scFv C21 binding site but this orientationresulted in both Leu residues being exposed. Since exposed hydrophobicresidue orientation is not energetically suitable, we chose the exposed10Asp conformation as one of the most favorable binding modes forfurther minimization, but introduced a Ser residue at position 10. Incontrast to Asp, the Ser substituted peptide makes a contact with Tyr at49Leu of scFv C21 without much change in the Leu orientation.Intermolecular interaction analysis suggested that this Ser substitutedpeptide would bind more effectively with scFv C21 (Table 1). Thispossibility was also corroborated by Biacore analysis of the interactionof substituted peptides with scFv C21 (Table 2). Replacement of Asp withSer at position 10 (P3S10) had only a slight effect on the ability ofP3C21 to inhibit the reactivity of scFv C21 with Colo38 cells (FIG. 8).This finding suggests that at position 10, a polar substitution istolerated. The reduced inhibition may be due to the complex nature ofHWM-MAA-scFv C21-peptide interactions.

Example 5 HMW-MAA-Specific Monoclonal Antibodies for the Treatment ofCancer

This example describes the use of HMW-MAA-specific human monoclonalantibodies for the treatment of cancers that exhibit overexpression ofHMW-MAA (referred to herein as a “HMW-MAA-positive” cancer), including,but not limited to melanoma, breast cancer, prostate cancer and squamouscell carcinoma. Patients diagnosed with a HMW-MAA-positive cancer can betreated according to standard procedures in the art. Generally,treatment options include surgery, radiation therapy, chemotherapy,immunotherapy or interferon therapy.

In this example, patients diagnosed with a HMW-MAA-positive melanoma areadministered an immunoconjugate comprising a HMW-MAA-specific humanmonoclonal antibody linked to Pseudomonas exotoxin (PE). Preparation ofPE immunoconjugates has been described (see, for example, U.S. Pat. No.7,081,518 and U.S. Pre-Grant Publication No. 2005/0214304, which areherein incorporated by reference). In some patients, the immunoconjugateis administered by intravenous bolus injection every other day for atotal of three to six doses. In other patients, the immunoconjugate isadministered by continuous intravenous infusion over the course of tendays. The dose of immunoconjugate administered to a patient variesdepending on the weight and gender of the patient, and mode and timecourse of administration. Following treatment, patients are evaluatedfor cancer progression (including tumor growth and metastasis) and otherclinical signs of illness. Patients can be treated with theimmunoconjugate alone, or in combination with one or more standardcancer treatments. For example, a patient that has undergone surgery toremove the melanoma can subsequently be treated with theimmunoconjugate.

Example 6 Additional Materials and Methods

Mice:

Female SCID/BALB/c mice (C.B-Igh-1^(b)IcrTac-Prkdcscid, 6-8 weeks old)were purchased from NCl or Taconic Farms, Inc. Experiments for thisstudy were approved by the Institutional Animal Care and Use Committee.

Cell Lines:

The human tumor cell lines were maintained in RPMI 1640 mediumsupplemented with 10% FCS.

Antibodies:

scFv-Fc C21 and scFv-Fc 119, which was used as an isotype control werepurified from mouse ascites by sequential ammonium sulphate and caprylicacid precipitation and Protein A column PE-anti-human IgG antibodies waspurchased from Jackson ImmunoResearch Laboratories, Inc. Specificantibodies for signaling molecules were purchased commercially for: PKCα(Sigma); FAK and phosphorylated FAK (Tyr397) (BD Bioscience); PDK andphosphorylated PDK1(ser241), Akt and phosphorylated Akt (Ser473),phosphorylated-Src (Tyr416), Erk1/2 and phosphorylated 44/42 Erk1/2,(Thr202/Tyr204), anti-β-catenin and phosphorylated-Histone H3(Ser10)(Cell signaling technology).

Immunohistochemistry, Phospho-Histone H3 (Ser10):

FFPE of lung sections were stained with anti-p-Histone3 antibodyaccording to the manufacturer's protocol.

Flow Cytometry Analysis:

Tumor cell preparations from cultured cell lines were stained byCSPG4-specific scFv-Fc C21 for 30 min, washed twice with PBS, andincubated for 30 min with PE-labeled anti-human IgG antibody. After 3washes, cells were analyzed by flow cytometry. scFv-Fc 119 was used asisotype control.

Western Blotting:

Western blot assay for signaling related proteins was performed usingstandard techniques on the lysates prepared from: i) cultured MV3 cells(2×10⁴ cells/well in a 96-well plate) serum starved for 72 hours,incubated with either scFv-Fc C21 (50 μg/ml), isotype matched control(50 μg/ml) or PBS for an additional 48 hrs, and lysed in lysis buffer(10 mM Tris-HC, 1% NP40, 1 mM EDTA, 0.1% BSA, 150 mM NaCl, 1/50 ofprotease inhibitor cocktail (Calbiochem); and ii) snap frozen surgicallyremoved xenografts homogenized before and after adding an ice-cold RIPAbuffer (Thermo Scientific) containing 1/50 of protease inhibitorcocktail (Calbiochem). After vortexing for 60 seconds, samples wereice-cooled for 45 minutes. Insoluble material was removed from tissuelysates by centrifugation at 13,000 rpm for 30 minutes at 4° C. Proteinconcentration was measured by Bradford reagent (Bio-Rad). Equal amountsof clarified lysate protein were separated by sodium dodecylsulfate—polyacrylamide gel electrophoresis and transferred onto 0.45-μm(pore size) PVDF (Millipore). After blocking with 5% nonfat dry milkplus 2% BSA for 2 hour at room temperature, membranes were probed withanti-PKCα, anti-phosphorylated FAK, anti-FAK, anti-PDK,anti-phosphorylated PDK1(ser241), anti-phosphorylated Akt, anti-Akt,anti-phosphorylated Src (Tyr416), anti-anti-Erk1/2, anti-phosphorylatedErk1/2 (Thr202/Tyr204), anti-β-Catenin, and HLA class I antibody HC-10(0.2 μg/ml) or anto-calnexin mAb To5 (0.2 μg/ml) overnight at 4° C., andthen with secondary antibody. Bands were visualized with the EnhancedChemiluminescence System (GE Life Science), and band density was readwith the FOTO/ANALYST® Investigator Eclipse system (FotodyneIncorporate) and quantified with TOTALLAB™ TL100 software (NonlinearDynamics). HLA class I or calnexin was used as the loading controls.

Cell Growth and Migration:

For cell growth assay, cells (5×10⁴/well) were serum starved for 48 hrsand then seeded in a 96-well plate containing 4 times diluted MATRIGEL™(growth factor-reduced MATRIGEL™-CB-40230, BD Biosciences) and eitherscFv-Fc C21 (0.5 mg/ml), control scFv-Fc 119 (0.5 mg/ml) or PBS in serumfree RPMI 1640 medium (total volume 200 μl/well) and cultured at 37° C.in a 5% CO₂ atmosphere for 6 days. For migration assay, cells(5×10⁴/well) were serum starved 48 hours and then seeded in a24-transwell plate (24-well insert, pore size 8 μm; BD Biosciences) withscFv-Fc C21 (0.5 mg/ml), control scFv-Fc 119 (0.5 mg/ml) or PBS. Cellsmigrated toward to serum-free RPMI1640 medium containing 10 μg/mlfibronectin. After 48 hours, migrated cells were stained with HEMA 3stain set, taken picture and counted under a Zeiss Inverted FluorescenceMicroscope (AxioVision Software). Mean of six independent high powerfield (200×) are shown as columns. All above experiments were performedin triplicates.

Experimental Lung Metastasis and Antibody Administration:

SCID mice were intravenously injected with cells MV3 and treated withscFc-Fc C21, as indicated in FIG. 16

Spontaneous Lung Metastasis and Treatment Experiments:

MV3 cells were implanted subcutaneously into SCID mice. Primary tumorswere surgically removed while the mice were fully anesthetized usingKetamine (5 mg/kg, intraperitoneally (i.p.)) and continuous isofluraneinhalation. After the surgery, the mice were given the analgesic drugKetoprofen (5 mg/kg, subcuntaneous (s.c.) administration) daily for 3days. scFc-Fc C21 were administered as indicated in FIG. 18.

Example 7 Reactivity of scFv-Fc C21 with a Panel of Human Tumor CellLines

Flow analysis has shown that scFv-Fc C21 stained specifically the cellsurface of a panel of human cultured cell lines, including melanoma celllines MDA-MB-435, MV3 and WM1158; glioma cell lines LN444 and A1207 andhead and neck cancer cell line PCI30 and PCI13. CSPG4⁻ melanoma cellline M14 was used as a negative control and M14/CSPG4, which is a CSPG4transfectant of M14 cell line, was used as a positive control (see FIGS.14-15).

Example 8 Inhibition by scFv-Fc C21 of Tumor Cell Growth, and Migration

scFv-Fc21 inhibited tumor cell growth and migration in vitro (FIGS.22-23). The methods are described above in Example 6.

Example 9 Inhibition In Vitro of Multiple Signaling Pathways, which areImportant to Tumor Cell Growth, Survival and Metastasis

scFv-Fc C21 treated MV3 cells had a decreased level of Protein kinase Calpha (PKCα) and FAK. Moreover, scFv-Fc C21 treatment inhibited thephosphorylation/activation of FAK, Erk1/2, PDK1 (upstream of Akt) andAkt. The decreased Akt activation was accompanied with an increase inphosphorylated/activated PTEN, a negative regulator of PI3K/Akt pathwaysignaling (see FIGS. 24-26).

Example 10 Inhibition by scFc-Fc C21 of Human Melanoma EstablishedExperimental Metastasis In Vivo

scFc-Fc C21 inhibited significantly experimental metastases of MV3 cells(FIG. 16). Metastatic lesions were also evaluated for the rate of tumorcell proliferation using the surrogate marker p-Histone H3. Thetreatment of scFc-Fc C21 significantly reduced the number of mitoticcells (FIG. 17).

Example 11 Inhibition by scFc-Fc C21 of Human Melanoma Post-SurgeryRecurrence and Spontaneous Metastasis In Vivo

The ability of scFc-Fc C21 to inhibit tumor recurrence and spontaneousmetastasis of MV3 tumors in SCID mice was tested following surgicalremoval of primary tumors. Mice treated with scFc-Fc C21 hadsignificantly lower rate of tumor recurrence and lower levels ofspontaneous lung metastases than those receiving a control scFv-Fc(FIGS. 18-20). Additionally, primary tumors surgically removed from micetreated with scFc-Fc C21 were also evaluated for the activation ofspecific signal transduction pathways associated with melanoma growthand progression. The activation of PKC-α and Src signaling pathways wasmarkedly reduced in the primary tumors removed from the mice treatedwith scFv-Fc C21 as compared to those removed from the mice treated withthe control scFv-Fc 119 (FIG. 21).

This disclosure provides fully human antibodies specific for HMW-MAA.The disclosure further provides methods of treating or detecting cancersassociated with expression of HMW-MAA, and methods of treatment usingthese antibodies. It will be apparent that the precise details of themethods described may be varied or modified without departing from thespirit of the described disclosure. We claim all such modifications andvariations that fall within the scope and spirit of the claims below.

The invention claimed is:
 1. An isolated human monoclonal antibody orantigen-binding fragment thereof, wherein the heavy chain variableregion of the antibody or antigen-binding fragment thereof comprisesamino acids 27-38 of SEQ ID NO: 5, amino acids 56-65 of SEQ ID NO: 5,and amino acids 105-115 of SEQ ID NO: 5; and the light chain variableregion of the antibody or antigen-binding fragment thereof comprisesamino acids 27-38 of SEQ ID NO: 6, amino acids 56-65 of SEQ ID NO: 6,and amino acids 105-110 of SEQ ID NO: 6, and wherein the antibodyspecifically binds HMW-MAA.
 2. The isolated human monoclonal antibody orantigen-binding fragment thereof of claim 1, wherein the heavy chainvariable region comprises SEQ ID NO:
 5. 3. The isolated human monoclonalantibody or antigen-binding fragment thereof of claim 1, wherein thelight chain variable region comprises SEQ ID NO:
 6. 4. The isolatedhuman monoclonal antibody or antigen-binding fragment thereof of claim1, comprising the heavy chain variable region of SEQ ID NO: 5 and thelight chain variable region of SEQ ID NO:
 6. 5. The isolated humanmonoclonal antibody or antigen-binding fragment thereof of claim 1,wherein the antigen-binding fragment is a Fab, a Fab′, a F(ab)′₂, asingle chain Fv (scFv), or a disulfide stabilized Fv (dsFv).
 6. Theisolated antigen-binding fragment of claim 5, wherein theantigen-binding fragment is a scFv.
 7. The isolated human monoclonalantibody of claim 1, wherein the antibody is an IgG.
 8. The isolatedhuman monoclonal antibody or antigen-binding fragment thereof of claim 1wherein the antibody or antigen-binding fragment thereof is labeled. 9.A composition comprising the antibody or antigen-binding fragmentthereof of claim 1, and a pharmaceutically acceptable carrier.
 10. Anisolated immunoconjugate comprising the human monoclonal antibody orantigen-binding fragment thereof of claim
 1. 11. The isolatedimmunoconjugate of claim 10, comprising Pseudomonas exotoxin (PE), or acytotoxic variant or fragment thereof.
 12. A composition comprising theisolated immunoconjugate of claim 10 and a pharmaceutically acceptablecarrier.